《生物化学原理》英文版（三）：8

M etabolism is a highly coordinated cellular activity in which many multienzyme systems (metabolic pathways) cooperate to (1) obtain chemical energy by capturing solar energy or degrading energy-rich nutrients from the environment; (2) convert nutrient molecules into the cell’s own characteristic molecules, including precursors of macromolecules; (3) polymerize mono- meric precursors into macromolecules: proteins, nucleic acids, and polysaccharides; and (4) synthesize and degrade biomolecules required for specialized cellular functions, such as membrane lipids, intracellular mes- sengers, and pigments. Although metabolism embraces hundreds of differ- ent enzyme-catalyzed reactions, our major concern in Part II is the central metabolic pathways, which are few in number and remarkably similar in all forms of life. Living organisms can be divided into two large groups according to the chemical form in which they obtain carbon from the environment. Autotrophs (such as photosynthetic bacteria and vascular plants) can use carbon dioxide from the atmosphere as their sole source of carbon, from which they construct all their carbon- containing biomolecules (see Fig. 1–5). Some auto- trophic organisms, such as cyanobacteria, can also use atmospheric nitrogen to generate all their nitrogenous components. Heterotrophs cannot use atmospheric carbon dioxide and must obtain carbon from their en- vironment in the form of relatively complex organic mol- ecules such as glucose. Multicellular animals and most microorganisms are heterotrophic. Autotrophic cells and organisms are relatively self-sufficient, whereas het- erotrophic cells and organisms, with their requirements for carbon in more complex forms, must subsist on the products of other organisms. Many autotrophic organisms are photosynthetic and obtain their energy from sunlight, whereas het- erotrophic organisms obtain their energy from the degradation of organic nutrients produced by auto- trophs. In our biosphere, autotrophs and heterotrophs live together in a vast, interdependent cycle in which autotrophic organisms use atmospheric carbon dioxide to build their organic biomolecules, some of them gen- erating oxygen from water in the process. Heterotrophs in turn use the organic products of autotrophs as nu- trients and return carbon dioxide to the atmosphere. Some of the oxidation reactions that produce carbon dioxide also consume oxygen, converting it to water. Thus carbon, oxygen, and water are constantly cycled between the heterotrophic and autotrophic worlds, with PART BIOENERGETICS AND METABOLISM II 13 Principles of Bioenergetics 480 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway 521 15 Principles of Metabolic Regulation, Illustrated with the Metabolism of Glucose and Glycogen 560 16 The Citric Acid Cycle 601 17 Fatty Acid Catabolism 631 18 Amino Acid Oxidation and the Production of Urea 666 19 Oxidative Phosphorylation and Photophosphorylation 700 20 Carbohydrate Biosynthesis in Plants and Bacteria 761 21 Lipid Biosynthesis 797 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules 843 23 Integration and Hormonal Regulation of Mammalian Metabolism 891 481 solar energy as the driving force for this global process (Fig. 1). All living organisms also require a source of nitro- gen, which is necessary for the synthesis of amino acids, nucleotides, and other compounds. Plants can generally use either ammonia or nitrate as their sole source of ni- trogen, but vertebrates must obtain nitrogen in the form of amino acids or other organic compounds. Only a few organisms—the cyanobacteria and many species of soil bacteria that live symbiotically on the roots of some plants—are capable of converting (“fixing”) atmos- pheric nitrogen (N 2 ) into ammonia. Other bacteria (the nitrifying bacteria) oxidize ammonia to nitrites and ni- trates; yet others convert nitrate to N 2 . Thus, in addi- tion to the global carbon and oxygen cycle, a nitrogen cycle operates in the biosphere, turning over huge amounts of nitrogen (Fig. 2). The cycling of carbon, oxy- gen, and nitrogen, which ultimately involves all species, depends on a proper balance between the activities of the producers (autotrophs) and consumers (het- erotrophs) in our biosphere. These cycles of matter are driven by an enormous flow of energy into and through the biosphere, begin- ning with the capture of solar energy by photosynthetic organisms and use of this energy to generate energy- rich carbohydrates and other organic nutrients; these nutrients are then used as energy sources by het- erotrophic organisms. In metabolic processes, and in all energy transformations, there is a loss of useful energy (free energy) and an inevitable increase in the amount of unusable energy (heat and entropy). In contrast to the cycling of matter, therefore, energy flows one way through the biosphere; organisms cannot regenerate useful energy from energy dissipated as heat and entropy. Carbon, oxygen, and nitrogen recycle continu- ously, but energy is constantly transformed into unus- able forms such as heat. Metabolism, the sum of all the chemical transfor- mations taking place in a cell or organism, occurs through a series of enzyme-catalyzed reactions that con- stitute metabolic pathways. Each of the consecutive steps in a metabolic pathway brings about a specific, small chemical change, usually the removal, transfer, or addition of a particular atom or functional group. The precursor is converted into a product through a series of metabolic intermediates called metabolites. The term intermediary metabolism is often applied to the combined activities of all the metabolic pathways that interconvert precursors, metabolites, and products of low molecular weight (generally, M r H110211,000). Catabolism is the degradative phase of metabolism in which organic nutrient molecules (carbohydrates, fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO 2 , NH 3 ). Catabolic pathways release energy, some of which is conserved in the formation of ATP and reduced electron carriers (NADH, NADPH, and FADH 2 ); the rest is lost as heat. In anabolism, also called biosynthesis, small, simple precursors are built up into larger and more complex Part II Bioenergetics and Metabolism482 Heterotrophs O 2 H 2 O Photosynthetic autotrophs O r g a n ic pro d u c t s C O 2 FIGURE 1 Cycling of carbon dioxide and oxygen between the auto- trophic (photosynthetic) and heterotrophic domains in the biosphere. The flow of mass through this cycle is enormous; about 4 H11003 10 11 met- ric tons of carbon are turned over in the biosphere annually. Plants Nitrates, nitrites Nitrifying bacteria Denitrifying bacteria Animals Amino acids Ammonia Nitrogen- fixing bacteria Atmospheric N 2 FIGURE 2 Cycling of nitrogen in the biosphere. Gaseous nitrogen (N 2 ) makes up 80% of the earth’s atmosphere. molecules, including lipids, polysaccharides, proteins, and nucleic acids. Anabolic reactions require an input of energy, generally in the form of the phosphoryl group transfer potential of ATP and the reducing power of NADH, NADPH, and FADH 2 (Fig. 3). Some metabolic pathways are linear, and some are branched, yielding multiple useful end products from a single precursor or converting several starting materi- als into a single product. In general, catabolic pathways are convergent and anabolic pathways divergent (Fig. 4). Some pathways are cyclic: one starting component of the pathway is regenerated in a series of reactions that converts another starting component into a prod- uct. We shall see examples of each type of pathway in the following chapters. Most cells have the enzymes to carry out both the degradation and the synthesis of the important cate- gories of biomolecules—fatty acids, for example. The simultaneous synthesis and degradation of fatty acids would be wasteful, however, and this is prevented by reciprocally regulating the anabolic and catabolic reac- tion sequences: when one sequence is active, the other is suppressed. Such regulation could not occur if ana- bolic and catabolic pathways were catalyzed by exactly the same set of enzymes, operating in one direction for anabolism, the opposite direction for catabolism: inhi- bition of an enzyme involved in catabolism would also inhibit the reaction sequence in the anabolic direction. Catabolic and anabolic pathways that connect the same two end points (glucose nnpyruvate and pyruvate nnglucose, for example) may employ many of the same enzymes, but invariably at least one of the steps is catalyzed by different enzymes in the catabolic and anabolic directions, and these enzymes are the sites of separate regulation. Moreover, for both anabolic and catabolic pathways to be essentially irreversible, the re- actions unique to each direction must include at least one that is thermodynamically very favorable—in other words, a reaction for which the reverse reaction is very unfavorable. As a further contribution to the separate regulation of catabolic and anabolic reaction sequences, paired catabolic and anabolic pathways commonly take place in different cellular compartments: for example, fatty acid catabolism in mitochondria, fatty acid syn- thesis in the cytosol. The concentrations of intermedi- ates, enzymes, and regulators can be maintained at different levels in these different compartments. Be- cause metabolic pathways are subject to kinetic con- trol by substrate concentration, separate pools of anabolic and catabolic intermediates also contribute to the control of metabolic rates. Devices that separate anabolic and catabolic processes will be of particular interest in our discussions of metabolism. Metabolic pathways are regulated at several levels, from within the cell and from outside. The most imme- diate regulation is by the availability of substrate; when the intracellular concentration of an enzyme’s substrate is near or below K m (as is commonly the case), the rate of the reaction depends strongly upon substrate con- centration (see Fig. 6–11). A second type of rapid con- trol from within is allosteric regulation (p. 225) by a metabolic intermediate or coenzyme—an amino acid or ATP, for example—that signals the cell’s internal meta- bolic state. When the cell contains an amount of, say, aspartate sufficient for its immediate needs, or when the cellular level of ATP indicates that further fuel con- sumption is unnecessary at the moment, these signals allosterically inhibit the activity of one or more enzymes in the relevant pathway. In multicellular organisms the metabolic activities of different tissues are regulated and integrated by growth factors and hormones that act from outside the cell. In some cases this regulation occurs virtually instantaneously (sometimes in less than a mil- lisecond) through changes in the levels of intracellular Part II Bioenergetics and Metabolism 483 Precursor molecules Amino acids Sugars Fatty acids Nitrogenous bases Energy- containing nutrients Carbohydrates Fats Proteins Anabolism ATP NADH NADPH FADH 2 Catabolism Chemical energy ADP H11001 HPO 2H11002 NAD H11001 NADP H11001 FAD 4 Cell macromolecules Proteins Polysaccharides Lipids Nucleic acids Energy- depleted end products CO 2 H 2 O NH 3 FIGURE 3 Energy relationships between catabolic and anabolic pathways. Catabolic pathways deliver chemical energy in the form of ATP, NADH, NADPH, and FADH 2 . These energy carriers are used in anabolic pathways to convert small precursor molecules into cell macromolecules. messengers that modify the activity of existing enzyme molecules by allosteric mechanisms or by covalent mod- ification such as phosphorylation. In other cases, the ex- tracellular signal changes the cellular concentration of an enzyme by altering the rate of its synthesis or degra- dation, so the effect is seen only after minutes or hours. The number of metabolic transformations taking place in a typical cell can seem overwhelming to a be- ginning student. Most cells have the capacity to carry out thousands of specific, enzyme-catalyzed reactions: for example, transformation of a simple nutrient such as glucose into amino acids, nucleotides, or lipids; ex- traction of energy from fuels by oxidation; or polymer- ization of monomeric subunits into macromolecules. Fortunately for the student of biochemistry, there are patterns within this multitude of reactions; you do not need to learn all these reactions to comprehend the molecular logic of biochemistry. Most of the reactions in living cells fall into one of five general categories: (1) oxidation-reductions; (2) reactions that make or break carbon–carbon bonds; (3) internal rearrangements, isomerizations, and eliminations; (4) group transfers; and (5) free radical reactions. Reactions within each general category usually proceed by a limited set of mechanisms and often employ characteristic cofactors. Before reviewing the five main reaction classes of biochemistry, let’s consider two basic chemical princi- ples. First, a covalent bond consists of a shared pair of electrons, and the bond can be broken in two general ways (Fig. 5). In homolytic cleavage, each atom leaves the bond as a radical, carrying one of the two electrons (now unpaired) that held the bonded atoms together. In the more common, heterolytic cleavage, one atom re- tains both bonding electrons. The species generated when COC and COH bonds are cleaved are illustrated in Figure 5. Carbanions, carbocations, and hydride ions are highly unstable; this instability shapes the chemistry of these ions, as described further below. The second chemical principle of interest here is that many biochemical reactions involve interactions between nucleophiles (functional groups rich in electrons and capable of donating them) and electrophiles (electron- deficient functional groups that seek electrons). Nucle- ophiles combine with, and give up electrons to, elec- trophiles. Common nucleophiles and electrophiles are listed in Figure 6–21. Note that a carbon atom can act as either a nucleophile or an electrophile, depending on which bonds and functional groups surround it. We now consider the five main reaction classes you will encounter in upcoming chapters. Part II Bioenergetics and Metabolism484 Rubber Bile acids Steroid hormones (a) Converging catabolism (b) Diverging anabolism Oxaloacetate CO 2 CO 2 (c) Cyclic pathway Acetate (acetyl-CoA) Citrate PyruvateGlucoseGlycogen Phospholipids Alanine Fatty acids Leucine Phenyl- alanine Isoleucine Starch SerineSucrose Eicosanoids Phospholipids Carotenoid pigments Vitamin K Triacylglycerols Cholesteryl esters Triacylglycerols Mevalonate Isopentenyl- pyrophosphate Fatty acids Acetoacetyl-CoA CDP-diacylglycerol Cholesterol FIGURE 4 Three types of nonlinear metabolic pathways. (a) Con- verging, catabolic; (b) diverging, anabolic; and (c) cyclic, in which one of the starting materials (oxaloacetate in this case) is regenerated and reenters the pathway. Acetate, a key metabolic intermediate, is the breakdown product of a variety of fuels (a), serves as the precur- sor for an array of products (b), and is consumed in the catabolic path- way known as the citric acid cycle (c). 1. Oxidation-reduction reactions Carbon atoms encoun- tered in biochemistry can exist in five oxidation states, depending on the elements with which carbon shares electrons (Fig. 6). In many biological oxidations, a com- pound loses two electrons and two hydrogen ions (that is, two hydrogen atoms); these reactions are commonly called dehydrogenations and the enzymes that catalyze them are called dehydrogenases (Fig. 7). In some, but not all, biological oxidations, a carbon atom becomes co- valently bonded to an oxygen atom. The enzymes that catalyze these oxidations are generally called oxidases or, if the oxygen atom is derived directly from molecu- lar oxygen (O 2 ), oxygenases. Every oxidation must be accompanied by a reduc- tion, in which an electron acceptor acquires the electrons removed by oxidation. Oxidation reactions generally release energy (think of camp fires: the compounds in wood are oxidized by oxygen molecules in the air). Most living cells obtain the energy needed for cellular work by oxidizing metabolic fuels such as carbohydrates or fat; photosynthetic organisms can also trap and use the en- ergy of sunlight. The catabolic (energy-yielding) path- ways described in Chapters 14 through 19 are oxidative reaction sequences that result in the transfer of electrons from fuel molecules, through a series of electron carri- ers, to oxygen. The high affinity of O 2 for electrons makes the overall electron-transfer process highly exergonic, providing the energy that drives ATP synthesis—the central goal of catabolism. 2. Reactions that make or break carbon–carbon bonds Het- erolytic cleavage of a COC bond yields a carbanion and a carbocation (Fig. 5). Conversely, the formation of a COC bond involves the combination of a nucleophilic carbanion and an electrophilic carbocation. Groups with electronegative atoms play key roles in these reactions. Carbonyl groups are particularly important in the chem- ical transformations of metabolic pathways. As noted above, the carbon of a carbonyl group has a partial pos- itive charge due to the electron-withdrawing nature of the adjacent bonded oxygen, and thus is an electrophilic carbon. The presence of a carbonyl group can also facilitate the formation of a carbanion on an adjoining carbon, because the carbonyl group can delocalize elec- trons through resonance (Fig. 8a, b). The importance of a carbonyl group is evident in three major classes of reactions in which COC bonds are formed or broken (Fig 8c): aldol condensations (such as the aldolase reaction; see Fig. 14–5), Claisen condensations (as in the citrate synthase reaction; see Fig. 16–9), and Part II Bioenergetics and Metabolism 485 CC Carbon radicals C H11001 C H11002 CH ProtonCarbanion C H11001 H H11001 Heterolytic cleavage CH Carbon radical C H11001 H Homolytic cleavage CH HydrideCarbocation C H11001 H11001 H11002 CC CarbocationCarbanion C H11001 H11001 C H atom H11002 H FIGURE 5 Two mechanisms for cleavage of a COC or COH bond. In homolytic cleavages, each atom keeps one of the bonding elec- trons, resulting in the formation of carbon radicals (carbons having unpaired electrons) or uncharged hydrogen atoms. In heterolytic cleav- ages, one of the atoms retains both bonding electrons. This can result in the formation of carbanions, carbocations, protons, or hydride ions. CH 2 AlkaneCH 3 CH 2 CH 2 Alcohol Aldehyde (ketone) Carboxylic acid Carbon dioxide CH 2 OH O H(R) C CH 2 O OO OH C C FIGURE 6 The oxidation states of carbon in biomolecules. Each com- pound is formed by oxidation of the red carbon in the compound listed above it. Carbon dioxide is the most highly oxidized form of carbon found in living systems. FIGURE 7 An oxidation-reduction reaction. Shown here is the oxi- dation of lactate to pyruvate. In this dehydrogenation, two electrons and two hydrogen ions (the equivalent of two hydrogen atoms) are re- moved from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the reaction is catalyzed by lactate dehydrogenase and the elec- trons are transferred to a cofactor called nicotinamide adenine dinu- cleotide. This reaction is fully reversible; pyruvate can be reduced by electrons from the cofactor. In Chapter 13 we discuss the factors that determine the direction of a reaction. CH 3 Lactate Pyruvatelactate dehydrogenase CH 3 CH OH C C C O O O H11002 2H H11001 2e H11002 H11001 2H H11001 2e H11002 H11001 O O H11002 decarboxylations (as in the acetoacetate decarboxylase reaction; see Fig. 17–18). Entire metabolic pathways are organized around the introduction of a carbonyl group in a particular location so that a nearby carbon–carbon bond can be formed or cleaved. In some reactions, this role is played by an imine group or a specialized cofac- tor such as pyridoxal phosphate, rather than by a car- bonyl group. 3. Internal rearrangements, isomerizations, and eliminations Another common type of cellular reaction is an in- tramolecular rearrangement, in which redistribution of electrons results in isomerization, transposition of dou- ble bonds, or cis-trans rearrangements of double bonds. An example of isomerization is the formation of fruc- tose 6-phosphate from glucose 6-phosphate during sugar metabolism (Fig 9a; this reaction is discussed in detail in Chapter 14). Carbon-1 is reduced (from alde- hyde to alcohol) and C-2 is oxidized (from alcohol to ketone). Figure 9b shows the details of the electron movements that result in isomerization. A simple transposition of a CUC bond occurs dur- ing metabolism of the common fatty acid oleic acid (see Fig. 17–9), and you will encounter some spectacular ex- amples of double-bond repositioning in the synthesis of cholesterol (see Fig. 21–35). Elimination of water introduces a CUC bond be- tween two carbons that previously were saturated (as in the enolase reaction; see Fig. 6–23). Similar reactions can result in the elimination of alcohols and amines. 4. Group transfer reactions The transfer of acyl, glycosyl, and phosphoryl groups from one nucleophile to another is common in living cells. Acyl group transfer generally involves the addition of a nucleophile to the carbonyl carbon of an acyl group to form a tetrahedral interme- diate. The chymotrypsin reaction is one example of acyl group transfer (see Fig. 6–21). Glycosyl group transfers in- volve nucleophilic substitution at C-1 of a sugar ring, which is the central atom of an acetal. In principle, the substitution could proceed by an S N 1 or S N 2 path, as described for the enzyme lysozyme (see Fig. 6–25). Phosphoryl group transfers play a special role in metabolic pathways. A general theme in metabolism is the attachment of a good leaving group to a metabolic intermediate to “activate” the intermediate for subse- quent reaction. Among the better leaving groups in nucleophilic substitution reactions are inorganic or- thophosphate (the ionized form of H 3 PO 4 at neutral pH, a mixture of H 2 PO 4 H11002 and HPO 4 2H11002 , commonly abbreviated P i ) and inorganic pyrophosphate (P 2 O 7 4H11002 , abbreviated PP i ); esters and anhydrides of phosphoric acid are effectively activated for reaction. Nucleophilic substi- tution is made more favorable by the attachment of a phosphoryl group to an otherwise poor leaving group such as OOH. Nucleophilic substitutions in which the R C Tetrahedral intermediate O Y X R C O H11002 Y X R C O Y X H11002 RCC H H OH R 1 H 2 O H H C H 2 O H R C R 1 Part II Bioenergetics and Metabolism486 CC H11002 CC H11001 C H9254H11001 (a) (b) (c) O H9254H11002 O O H11002 H11002 R 1 C Aldol condensation C O R 2 H C R 3 R 4 O H H11001 R 1 C C O R 2 H C R 3 R 4 OH CoA-S C Claisen ester condensation C O H H C R 1 R 2 O H H11001 CoA-S C C O H H C R 1 R 2 OH RC Decarboxylation of a H9252-keto acid C O H H C O O H11002 H H11001 RCC O H H H CO 2 H11002 FIGURE 8 Carbon–carbon bond formation reactions. (a) The carbon atom of a carbonyl group is an electrophile by virtue of the electron- withdrawing capacity of the electronegative oxygen atom, which results in a resonance hybrid structure in which the carbon has a partial pos- itive charge. (b) Within a molecule, delocalization of electrons into a carbonyl group facilitates the transient formation of a carbanion on an adjacent carbon. (c) Some of the major reactions involved in the for- mation and breakage of COC bonds in biological systems. For both the aldol condensation and the Claisen condensation, a carbanion serves as nucleophile and the carbon of a carbonyl group serves as elec- trophile. The carbanion is stabilized in each case by another carbonyl at the carbon adjoining the carbanion carbon. In the decarboxylation reaction, a carbanion is formed on the carbon shaded blue as the CO 2 leaves. The reaction would not occur at an appreciable rate but for the stabilizing effect of the carbonyl adjacent to the carbanion car- bon. Wherever a carbanion is shown, a stabilizing resonance with the adjacent carbonyl, as shown in (a), is assumed. The formation of the carbanion is highly disfavored unless the stabilizing carbonyl group, or a group of similar function such as an imine, is present. phosphoryl group (OPO 3 2H11002 ) serves as a leaving group occur in hundreds of metabolic reactions. Phosphorus can form five covalent bonds. The con- ventional representation of P i (Fig. 10a), with three POO bonds and one PUO bond, is not an accurate pic- ture. In P i , four equivalent phosphorus–oxygen bonds share some double-bond character, and the anion has a tetrahedral structure (Fig. 10b). As oxygen is more elec- tronegative than phosphorus, the sharing of electrons is unequal: the central phosphorus bears a partial positive charge and can therefore act as an electrophile. In a very large number of metabolic reactions, a phosphoryl group (OPO 3 2H11002 ) is transferred from ATP to an alcohol (form- ing a phosphate ester) (Fig. 10c) or to a carboxylic acid (forming a mixed anhydride). When a nucleophile at- tacks the electrophilic phosphorus atom in ATP, a rela- tively stable pentacovalent structure is formed as a re- action intermediate (Fig. 10d). With departure of the leaving group (ADP), the transfer of a phosphoryl group is complete. The large family of enzymes that catalyze Part II Bioenergetics and Metabolism 487 H 1 C 2 C B 1 H OOH Glucose 6-phosphate B 2 H CC H OOH C OH H C H OH C H OH C H H O P O H11002 O O H11002 H 1 C 2 C OH O Fructose 6-phosphate Enediol intermediate H C OH H C H OH C H OH C H H O P O H11002 O O H11002 (a) (b) phosphohexose isomerase 1 B 1 abstracts a proton. 4 B 2 abstracts a proton, allowing the formation of a C 2 This allows the formation of a C double bond. 3 Electrons from carbonyl form an 5 An electron leaves the C the hydrogen ion donated by B 2 . C C O bond. C bond to form a O H bond with CH bond with the proton donated by B 1 . B 1 HH C H OO H C OH H C O B 1 B 2 B 2 rows represent the movement of bonding electrons from nucleophile (pink) to electrophile (blue). B 1 and B 2 are basic groups on the enzyme; they are capable of donating and accepting hydrogen ions (protons) as the reaction progresses. FIGURE 9 Isomerization and elimination reactions. (a) The conver- sion of glucose 6-phosphate to fructose 6-phosphate, a reaction of sugar metabolism catalyzed by phosphohexose isomerase. (b) This re- action proceeds through an enediol intermediate. The curved blue ar- O H11002 OP O H11002 O H11002 H11002 O O O H11002 P H11002 O O H11002 O H11002 P OO H11002 O H11002 H11002 O O H11002 PO O O O 3 H11002 PO (a) (b) O P O O O O O O P WZ (d) (c) Adenine Ribose O O P O P O H11002 HO R O H11002 PO O H11002 O H11002 O O Glucose ATP Adenine Ribose O O P O H11002 O P OO H11002 H11001 O H11002 P R O H11002 H11002 O OO ADP Glucose 6-phosphate, a phosphate ester OHZ H11005 R W H11005 ADP FIGURE 10 Alternative ways of showing the structure of inorganic orthophosphate. (a) In one (inadequate) representation, three oxygens are single-bonded to phosphorus, and the fourth is double-bonded, allowing the four different resonance structures shown. (b) The four resonance structures can be represented more accurately by showing all four phosphorus–oxygen bonds with some double-bond character; the hybrid orbitals so represented are arranged in a tetrahedron with P at its center. (c) When a nucleophile Z (in this case, the OOH on C-6 of glucose) attacks ATP, it displaces ADP (W). In this S N 2 reac- tion, a pentacovalent intermediate (d) forms transiently. phosphoryl group transfers with ATP as donor are called kinases (Greek kinein, “to move”). Hexokinase, for ex- ample, “moves” a phosphoryl group from ATP to glucose. Phosphoryl groups are not the only activators of this type. Thioalcohols (thiols), in which the oxygen atom of an alcohol is replaced with a sulfur atom, are also good leaving groups. Thiols activate carboxylic acids by forming thioesters (thiol esters) with them. We will dis- cuss a number of cases, including the reactions cat- alyzed by the fatty acyl transferases in lipid synthesis (see Fig. 21–2), in which nucleophilic substitution at the carbonyl carbon of a thioester results in transfer of the acyl group to another moiety. 5. Free radical reactions Once thought to be rare, the homolytic cleavage of covalent bonds to generate free radicals has now been found in a range of biochemical processes. Some examples are the reactions of methyl- malonyl-CoA mutase (see Box 17–2), ribonucleotide reductase (see Fig. 22–41), and DNA photolyase (see Fig. 25–25). We begin Part II with a discussion of the basic en- ergetic principles that govern all metabolism (Chapter 13). We then consider the major catabolic pathways by which cells obtain energy from the oxidation of various fuels (Chapters 14 through 19). Chapter 19 is the piv- otal point of our discussion of metabolism; it concerns chemiosmotic energy coupling, a universal mechanism in which a transmembrane electrochemical potential, produced either by substrate oxidation or by light ab- sorption, drives the synthesis of ATP. Chapters 20 through 22 describe the major anabolic pathways by which cells use the energy in ATP to pro- duce carbohydrates, lipids, amino acids, and nucleotides from simpler precursors. In Chapter 23 we step back from our detailed look at the metabolic pathways—as they occur in all organisms, from Escherichia coli to humans—and consider how they are regulated and in- tegrated in mammals by hormonal mechanisms. As we undertake our study of intermediary metab- olism, a final word. Keep in mind that the myriad re- actions described in these pages take place in, and play crucial roles in, living organisms. As you encounter each reaction and each pathway ask, What does this chemi- cal transformation do for the organism? How does this pathway interconnect with the other pathways operat- ing simultaneously in the same cell to produce the en- ergy and products required for cell maintenance and growth? How do the multilayered regulatory mecha- nisms cooperate to balance metabolic and energy in- puts and outputs, achieving the dynamic steady state of life? Studied with this perspective, metabolism pro- vides fascinating and revealing insights into life, with countless applications in medicine, agriculture, and biotechnology. Part II Bioenergetics and Metabolism488 chapter L iving cells and organisms must perform work to stay alive, to grow, and to reproduce. The ability to har- ness energy and to channel it into biological work is a fundamental property of all living organisms; it must have been acquired very early in cellular evolution. Mod- ern organisms carry out a remarkable variety of energy transductions, conversions of one form of energy to an- other. They use the chemical energy in fuels to bring about the synthesis of complex, highly ordered macro- molecules from simple precursors. They also convert the chemical energy of fuels into concentration gradients and electrical gradients, into motion and heat, and, in a few organisms such as fireflies and some deep-sea fish, into light. Photosynthetic organisms transduce light en- ergy into all these other forms of energy. The chemical mechanisms that underlie biological energy transductions have fascinated and challenged biologists for centuries. Antoine Lavoisier, before he lost his head in the French Revolution, recognized that an- imals somehow transform chemical fuels (foods) into heat and that this process of respiration is essential to life. He observed that ...in general, respiration is nothing but a slow com- bustion of carbon and hy- drogen, which is entirely similar to that which oc- curs in a lighted lamp or candle, and that, from this point of view, animals that respire are true com- bustible bodies that burn and consume themselves . . . One may say that this analogy between combustion and respiration has not escaped the notice of the poets, or rather the philosophers of antiquity, and which they had ex- pounded and interpreted. This fire stolen from heaven, this torch of Prometheus, does not only rep- resent an ingenious and poetic idea, it is a faithful picture of the operations of nature, at least for an- imals that breathe; one may therefore say, with the ancients, that the torch of life lights itself at the mo- ment the infant breathes for the first time, and it does not extinguish itself except at death. * In this century, biochemical studies have revealed much of the chemistry underlying that “torch of life.” Biological energy transductions obey the same physical laws that govern all other natural processes. It is there- fore essential for a student of biochemistry to under- stand these laws and how they apply to the flow of energy in the biosphere. In this chapter we first review the laws of thermodynamics and the quantitative rela- tionships among free energy, enthalpy, and entropy. We then describe the special role of ATP in biological PRINCIPLES OF BIOENERGETICS 13.1 Bioenergetics and Thermodynamics 490 13.2 Phosphoryl Group Transfers and ATP 496 13.3 Biological Oxidation-Reduction Reactions 507 The total energy of the universe is constant; the total entropy is continually increasing. —Rudolf Clausius, The Mechanical Theory of Heat with Its Applications to the Steam-Engine and to the Physical Properties of Bodies, 1865 (trans. 1867) The isomorphism of entropy and information establishes a link between the two forms of power: the power to do and the power to direct what is done. —Fran?ois Jacob, La logique du vivant: une histoire de l’hérédité (The Logic of Life: A History of Heredity), 1970 13 489 *From a memoir by Armand Seguin and Antoine Lavoisier, dated 1789, quoted in Lavoisier, A. (1862) Oeuvres de Lavoisier, Imprimerie Impériale, Paris. Antoine Lavoisier, 1743–1794 energy exchanges. Finally, we consider the importance of oxidation-reduction reactions in living cells, the en- ergetics of electron-transfer reactions, and the electron carriers commonly employed as cofactors of the en- zymes that catalyze these reactions. 13.1 Bioenergetics and Thermodynamics Bioenergetics is the quantitative study of the energy transductions that occur in living cells and of the nature and function of the chemical processes underlying these transductions. Although many of the principles of ther- modynamics have been introduced in earlier chapters and may be familiar to you, a review of the quantitative aspects of these principles is useful here. Biological Energy Transformations Obey the Laws of Thermodynamics Many quantitative observations made by physicists and chemists on the interconversion of different forms of energy led, in the nineteenth century, to the formula- tion of two fundamental laws of thermodynamics. The first law is the principle of the conservation of energy: for any physical or chemical change, the total amount of energy in the universe remains constant; energy may change form or it may be transported from one region to another, but it cannot be created or destroyed. The second law of thermodynamics, which can be stated in several forms, says that the universe always tends toward increasing disorder: in all natu- ral processes, the entropy of the universe increases. Living organisms consist of collections of molecules much more highly organized than the surrounding ma- terials from which they are constructed, and organisms maintain and produce order, seemingly oblivious to the second law of thermodynamics. But living organisms do not violate the second law; they operate strictly within it. To discuss the application of the second law to bio- logical systems, we must first define those systems and their surroundings. The reacting system is the collection of matter that is undergoing a particular chemical or physical process; it may be an organism, a cell, or two reacting com- pounds. The reacting system and its surroundings to- gether constitute the universe. In the laboratory, some chemical or physical processes can be carried out in iso- lated or closed systems, in which no material or energy is exchanged with the surroundings. Living cells and or- ganisms, however, are open systems, exchanging both material and energy with their surroundings; living sys- tems are never at equilibrium with their surroundings, and the constant transactions between system and sur- roundings explain how organisms can create order within themselves while operating within the second law of thermodynamics. In Chapter 1 (p. 23) we defined three thermody- namic quantities that describe the energy changes oc- curring in a chemical reaction: Gibbs free energy, G, expresses the amount of energy capable of doing work during a reaction at constant temperature and pressure. When a reaction proceeds with the release of free energy (that is, when the system changes so as to possess less free energy), the free-energy change, H9004G, has a negative value and the reaction is said to be exergonic. In endergonic reactions, the system gains free energy and H9004G is positive. Enthalpy, H, is the heat content of the reacting system. It reflects the number and kinds of chemical bonds in the reactants and products. When a chemical reaction releases heat, it is said to be exothermic; the heat content of the products is less than that of the reactants and H9004H has, by convention, a negative value. Reacting systems that take up heat from their surroundings are endothermic and have positive values of H9004H. Entropy, S, is a quantitative expression for the randomness or disorder in a system (see Box 1–3). When the products of a reaction are less complex and more disordered than the reactants, the reaction is said to proceed with a gain in entropy. The units of H9004G and H9004H are joules/mole or calories/mole (recall that 1 cal H11005 4.184 J); units of entropy are joules/mole H11080 Kelvin (J/mol H11080 K) (Table 13–1). Under the conditions existing in biological systems (including constant temperature and pressure), changes in free energy, enthalpy, and entropy are re- lated to each other quantitatively by the equation H9004G H11005H9004H H11002 T H9004S (13–1) Chapter 13 Principles of Bioenergetics490 in which H9004G is the change in Gibbs free energy of the reacting system, H9004H is the change in enthalpy of the system, T is the absolute temperature, and H9004S is the change in entropy of the system. By convention, H9004S has a positive sign when entropy increases and H9004H, as noted above, has a negative sign when heat is released by the system to its surroundings. Either of these conditions, which are typical of favorable processes, tend to make H9004G negative. In fact, H9004G of a spontaneously reacting sys- tem is always negative. The second law of thermodynamics states that the entropy of the universe increases during all chemical and physical processes, but it does not require that the entropy increase take place in the reacting system it- self. The order produced within cells as they grow and divide is more than compensated for by the disorder they create in their surroundings in the course of growth and division (see Box 1–3, case 2). In short, living or- ganisms preserve their internal order by taking from the surroundings free energy in the form of nutrients or sun- light, and returning to their surroundings an equal amount of energy as heat and entropy. Cells Require Sources of Free Energy Cells are isothermal systems—they function at essen- tially constant temperature (they also function at con- stant pressure). Heat flow is not a source of energy for cells, because heat can do work only as it passes to a zone or object at a lower temperature. The energy that cells can and must use is free energy, described by the Gibbs free-energy function G, which allows prediction of the direction of chemical reactions, their exact equi- librium position, and the amount of work they can in theory perform at constant temperature and pressure. Heterotrophic cells acquire free energy from nutrient molecules, and photosynthetic cells acquire it from ab- sorbed solar radiation. Both kinds of cells transform this free energy into ATP and other energy-rich compounds capable of providing energy for biological work at con- stant temperature. The Standard Free-Energy Change Is Directly Related to the Equilibrium Constant The composition of a reacting system (a mixture of chemical reactants and products) tends to continue changing until equilibrium is reached. At the equilibrium concentration of reactants and products, the rates of the forward and reverse reactions are exactly equal and no further net change occurs in the system. The concen- trations of reactants and products at equilibrium define the equilibrium constant, K eq (p. 26). In the general reaction aA H11001 bB cC H11001 dD, where a, b, c, and d are the number of molecules of A, B, C, and D par- ticipating, the equilibrium constant is given by K eq H11005 H5007 [ [ C A ] ] c a [ [ D B ] ] d b H5007 (13–2) where [A], [B], [C], and [D] are the molar concentrations of the reaction components at the point of equilibrium. When a reacting system is not at equilibrium, the tendency to move toward equilibrium represents a driv- ing force, the magnitude of which can be expressed as the free-energy change for the reaction, H9004G. Under stan- dard conditions (298 K H11005 25 H11034C), when reactants and products are initially present at 1 M concentrations or, for gases, at partial pressures of 101.3 kilopascals (kPa), or 1 atm, the force driving the system toward equilib- rium is defined as the standard free-energy change, H9004GH11034. By this definition, the standard state for reactions that involve hydrogen ions is [H H11001 ] H11005 1 M, or pH 0. Most bio- chemical reactions, however, occur in well-buffered aqueous solutions near pH 7; both the pH and the con- centration of water (55.5 M) are essentially constant. For convenience of calculations, biochemists therefore define a different standard state, in which the concen- tration of H H11001 is 10 H110027 M (pH 7) and that of water is 55.5 M; for reactions that involve Mg 2H11001 (including most in which ATP is a reactant), its concentration in solu- tion is commonly taken to be constant at 1 mM. Physi- cal constants based on this biochemical standard state are called standard transformed constants and are written with a prime (such as H9004GH11032H11034 and KH11032 eq ) to distin- guish them from the untransformed constants used by chemists and physicists. (Notice that most other text- books use the symbol H9004GH11034H11032 rather than H9004GH11032H11034. Our use of H9004GH11032H11034, recommended by an international committee of chemists and biochemists, is intended to emphasize that the transformed free energy GH11032 is the criterion for equi- librium.) By convention, when H 2 O, H H11001 , and/or Mg 2H11001 are reactants or products, their concentrations are not included in equations such as Equation 13–2 but are in- stead incorporated into the constants KH11032 eq and H9004GH11032H11034. z y 13.1 Bioenergetics and Thermodynamics 491 Boltzmann constant, k H11005 1.381 H11003 10 H1100223 J/K Avogadro’s number, N H11005 6.022 H11003 10 23 mol H110021 Faraday constant, H11005 96,480 J/V H11080 mol Gas constant, R H11005 8.315 J/mol H11080 K (H11005 1.987 cal/mol H11080 K) Units of H9004G and H9004H are J/mol (or cal/mol) Units of H9004S are J/mol H11554 K (or cal/mol H11554 K) 1 cal H11005 4.184 J Units of absolute temperature, T, are Kelvin, K 25 H11034C H11005 298 K At 25 H11034C, RT H11005 2.479 kJ/mol (H11005 0.592 kcal/mol) TABLE 13–1 Some Physical Constants and Units Used in Thermodynamics Just as KH11032 eq is a physical constant characteristic for each reaction, so too is H9004GH11032H11034 a constant. As we noted in Chapter 6, there is a simple relationship between KH11032 eq and H9004GH11032H11034: H9004GH11032H11034 H11005 H11002RT ln KH11032 eq The standard free-energy change of a chemical re- action is simply an alternative mathematical way of expressing its equilibrium constant. Table 13–2 shows the relationship between H9004GH11032H11034 and KH11032 eq . If the equilibrium constant for a given chemical reaction is 1.0, the standard free-energy change of that reaction is 0.0 (the natural logarithm of 1.0 is zero). If KH11032 eq of a reac- tion is greater than 1.0, its H9004GH11032H11034 is negative. If KH11032 eq is less than 1.0, H9004GH11032H11034 is positive. Because the relationship be- tween H9004GH11032H11034 and KH11032 eq is exponential, relatively small changes in H9004GH11032H11034 correspond to large changes in KH11032 eq . It may be helpful to think of the standard free- energy change in another way. H9004GH11032H11034 is the difference be- tween the free-energy content of the products and the free-energy content of the reactants, under standard conditions. When H9004GH11032H11034 is negative, the products contain less free energy than the reactants and the reaction will proceed spontaneously under standard conditions; all chemical reactions tend to go in the direction that re- sults in a decrease in the free energy of the system. A positive value of H9004GH11032H11034 means that the products of the reaction contain more free energy than the reactants, and this reaction will tend to go in the reverse direction if we start with 1.0 M concentrations of all components (standard conditions). Table 13–3 summarizes these points. As an example, let’s make a simple calculation of the standard free-energy change of the reaction cat- alyzed by the enzyme phosphoglucomutase: Glucose 1-phosphate 34 glucose 6-phosphate Chemical analysis shows that whether we start with, say, 20 mM glucose 1-phosphate (but no glucose 6-phosphate) or with 20 mM glucose 6-phosphate (but no glucose 1-phosphate), the final equilibrium mixture at 25 H11034C and pH 7.0 will be the same: 1 mM glucose 1-phosphate and 19 mM glucose 6-phosphate. (Remember that enzymes do not affect the point of equilibrium of a reaction; they merely hasten its attainment.) From these data we can calculate the equilibrium constant: KH11032 eq H11005H11005H5007 1 1 9 m m M M H5007 H11005 19 From this value of KH11032 eq we can calculate the standard free-energy change: H9004GH11032H11034 H11005 H11002RT ln KH11032 eq H11005H11002(8.315 J/mol H11554 K)(298 K)(ln 19) H11005H110027.3 kJ/mol Because the standard free-energy change is negative, when the reaction starts with 1.0 M glucose 1-phosphate and 1.0 M glucose 6-phosphate, the conversion of glu- cose 1-phosphate to glucose 6-phosphate proceeds with a loss (release) of free energy. For the reverse reaction (the conversion of glucose 6-phosphate to glucose 1-phosphate), H9004GH11032H11034 has the same magnitude but the op- posite sign. Table 13–4 gives the standard free-energy changes for some representative chemical reactions. Note that hydrolysis of simple esters, amides, peptides, and gly- cosides, as well as rearrangements and eliminations, proceed with relatively small standard free-energy changes, whereas hydrolysis of acid anhydrides is ac- companied by relatively large decreases in standard free energy. The complete oxidation of organic compounds such as glucose or palmitate to CO 2 and H 2 O, which in cells requires many steps, results in very large decreases in standard free energy. However, standard free-energy [glucose 6-phosphate] H5007H5007H5007 [glucose 1-phosphate] Chapter 13 Principles of Bioenergetics492 H9004GH11032H11034 KH11032 eq (kJ/mol) (kcal/mol)* 10 3 H1100217.1 H110024.1 10 2 H1100211.4 H110022.7 10 1 H110025.7 H110021.4 1 0.0 0.0 10 H110021 5.7 1.4 10 H110022 11.4 2.7 10 H110023 17.1 4.1 10 H110024 22.8 5.5 10 H110025 28.5 6.8 10 H110026 34.2 8.2 Relationship between the Equilibrium Constants and Standard Free-Energy Changes of Chemical Reactions TABLE 13–2 Starting with all components at 1 M, When KH11032 eq is . . . H9004GH11032H11034 is . . . the reaction . . . H110221.0 negative proceeds forward H110221.0 zero is at equilibrium H110211.0 positive proceeds in reverse Relationships among KH11032 eq , H9004GH11541H11543, and the Direction of Chemical Reactions under Standard Conditions TABLE 13–3 *Although joules and kilojoules are the standard units of energy and are used throughout this text, biochemists sometimes express H9004GH11032H11034 values in kilocalories per mole. We have therefore included values in both kilojoules and kilocalories in this table and in Tables 13–4 and 13–6. To convert kilojoules to kilocalories, divide the number of kilojoules by 4.184. changes such as those in Table 13–4 indicate how much free energy is available from a reaction under standard conditions. To describe the energy released under the conditions existing in cells, an expression for the actual free-energy change is essential. Actual Free-Energy Changes Depend on Reactant and Product Concentrations We must be careful to distinguish between two differ- ent quantities: the free-energy change, H9004G, and the stan- dard free-energy change, H9004GH11032H11034. Each chemical reaction has a characteristic standard free-energy change, which may be positive, negative, or zero, depending on the equilibrium constant of the reaction. The standard free- energy change tells us in which direction and how far a given reaction must go to reach equilibrium when the initial concentration of each component is 1.0 M, the pH is 7.0, the temperature is 25 H11034C, and the pressure is 101.3 kPa. Thus H9004GH11032H11034 is a constant: it has a character- istic, unchanging value for a given reaction. But the ac- tual free-energy change, H9004G, is a function of reactant and product concentrations and of the temperature pre- vailing during the reaction, which will not necessarily match the standard conditions as defined above. More- over, the H9004G of any reaction proceeding spontaneously toward its equilibrium is always negative, becomes less negative as the reaction proceeds, and is zero at the point of equilibrium, indicating that no more work can be done by the reaction. 13.1 Bioenergetics and Thermodynamics 493 H9004GH11032H11034 Reaction type (kJ/mol) (kcal/mol) Hydrolysis reactions Acid anhydrides Acetic anhydride H11001 H 2 O On 2 acetate H1100291.1 H1100221.8 ATP H11001 H 2 O 88n ADP H11001 P i H1100230.5 H110027.3 ATP H11001 H 2 O 88n AMP H11001 PP i H1100245.6 H1100210.9 PP i H11001 H 2 O 88n 2P i H1100219.2 H110024.6 UDP-glucose H11001 H 2 O 88n UMP H11001 glucose 1-phosphate H1100243.0 H1100210.3 Esters Ethyl acetate H11001 H 2 O 88n ethanol H11001 acetate H1100219.6 H110024.7 Glucose 6-phosphate H11001 H 2 O 88n glucose H11001 P i H1100213.8 H110023.3 Amides and peptides Glutamine H11001 H 2 O 88n glutamate H11001 NH 4 H11001 H1100214.2 H110023.4 Glycylglycine H11001 H 2 O 88n 2 glycine H110029.2 H110022.2 Glycosides Maltose H11001 H 2 O 88n 2 glucose H1100215.5 H110023.7 Lactose H11001 H 2 O 88n glucose H11001 galactose H1100215.9 H110023.8 Rearrangements Glucose 1-phosphate 88n glucose 6-phosphate H110027.3 H110021.7 Fructose 6-phosphate 88n glucose 6-phosphate H110021.7 H110020.4 Elimination of water Malate 88n fumarate H11001 H 2 O 3.1 0.8 Oxidations with molecular oxygen Glucose H11001 6O 2 88n 6CO 2 H11001 6H 2 O H110022,840 H11002686 Palmitate H11001 23O 2 88n 16CO 2 H11001 16H 2 O H110029,770 H110022,338 Standard Free-Energy Changes of Some Chemical Reactions at pH 7.0 and 25 H11034C (298 K) TABLE 13–4 DG and DGH11032H11034 for any reaction A H11001 B 34 C H11001 D are related by the equation H9004G H11005H9004GH11032H11034 H11001 RT ln H5007 [ [ C A ] ] [ [ D B] ] H5007 (13–3) in which the terms in red are those actually prevail- ing in the system under observation. The concentration terms in this equation express the effects commonly called mass action, and the term [C][D]/[A][B] is called the mass-action ratio, Q. As an example, let us sup- pose that the reaction A H11001 B 34 C H11001 D is taking place at the standard conditions of temperature (25 H11034C) and pressure (101.3 kPa) but that the concentrations of A, B, C, and D are not equal and none of the components is present at the standard concentration of 1.0 M. To de- termine the actual free-energy change, H9004G, under these nonstandard conditions of concentration as the reaction proceeds from left to right, we simply enter the actual concentrations of A, B, C, and D in Equation 13–3; the values of R, T, and H9004GH11032H11034 are the standard values. H9004G is negative and approaches zero as the reaction proceeds because the actual concentrations of A and B decrease and the concentrations of C and D increase. Notice that when a reaction is at equilibrium—when there is no force driving the reaction in either direction and H9004G is zero—Equation 13–3 reduces to 0 H11005H9004G H11005H9004GH11032H11034 H11001 RT ln H5007 [ [ C A ] ] e e q q [ [ D B] ] e e q q H5007 or H9004GH11032H11034 H11005 H11002RT ln KH11032 eq which is the equation relating the standard free-energy change and equilibrium constant given earlier. The criterion for spontaneity of a reaction is the value of H9004G, not H9004GH11032H11034. A reaction with a positive H9004GH11032H11034 can go in the forward direction if H9004G is negative. This is possible if the term RT ln ([products]/[reactants]) in Equation 13–3 is negative and has a larger absolute value than H9004GH11032H11034. For example, the immediate removal of the products of a reaction can keep the ratio [prod- ucts]/[reactants] well below 1, such that the term RT ln ([products]/[reactants]) has a large, negative value. H9004GH11032H11034 and H9004G are expressions of the maximum amount of free energy that a given reaction can theo- retically deliver—an amount of energy that could be realized only if a perfectly efficient device were avail- able to trap or harness it. Given that no such device is possible (some free energy is always lost to entropy dur- ing any process), the amount of work done by the re- action at constant temperature and pressure is always less than the theoretical amount. Another important point is that some thermody- namically favorable reactions (that is, reactions for which H9004GH11032H11034 is large and negative) do not occur at meas- urable rates. For example, combustion of firewood to CO 2 and H 2 O is very favorable thermodynamically, but firewood remains stable for years because the activation energy (see Figs 6–2 and 6–3) for the combustion re- action is higher than the energy available at room tem- perature. If the necessary activation energy is provided (with a lighted match, for example), combustion will be- gin, converting the wood to the more stable products CO 2 and H 2 O and releasing energy as heat and light. The heat released by this exothermic reaction provides the activation energy for combustion of neighboring regions of the firewood; the process is self-perpetuating. In living cells, reactions that would be extremely slow if uncatalyzed are caused to proceed, not by sup- plying additional heat but by lowering the activation en- ergy with an enzyme. An enzyme provides an alternative reaction pathway with a lower activation energy than the uncatalyzed reaction, so that at room temperature a large fraction of the substrate molecules have enough thermal energy to overcome the activation barrier, and the re- action rate increases dramatically. The free-energy change for a reaction is independent of the pathway by which the reaction occurs; it depends only on the nature and concentration of the initial reactants and the final products. Enzymes cannot, therefore, change equi- librium constants; but they can and do increase the rate at which a reaction proceeds in the direction dictated by thermodynamics. Standard Free-Energy Changes Are Additive In the case of two sequential chemical reactions, A 34 B and B 34 C, each reaction has its own equilibrium constant and each has its characteristic standard free- energy change, H9004G 1 H11032H11034 and H9004G 2 H11032H11034. As the two reactions are sequential, B cancels out to give the overall reaction A 34 C, which has its own equilibrium constant and thus its own standard free-energy change, H9004GH11032H11034 total . The H9004GH11032H11034 values of sequential chemical reactions are additive. For the overall reaction A 34 C, H9004GH11032H11034 total is the sum of the individual standard free-energy changes, H9004G 1 H11032H11034 and H9004G 2 H11032H11034, of the two reactions: H9004GH11032H11034 total H11005H9004G 1 H11032H11034 H11001 H9004G 2 H11032H11034. (1) A88nB H9004G 1 H11032H11034 (2) B88nC H9004G 2 H11032H11034 Sum: A88nC H9004G 1 H11032H11034 H11001 H9004G 2 H11032H11034 This principle of bioenergetics explains how a ther- modynamically unfavorable (endergonic) reaction can be driven in the forward direction by coupling it to a highly exergonic reaction through a common inter- mediate. For example, the synthesis of glucose 6- phosphate is the first step in the utilization of glucose by many organisms: Glucose H11001 P i 88n glucose 6-phosphate H11001 H 2 O H9004GH11032H11034 H11005 13.8 kJ/mol Chapter 13 Principles of Bioenergetics494 The positive value of H9004GH11032H11034 predicts that under standard conditions the reaction will tend not to proceed spon- taneously in the direction written. Another cellular re- action, the hydrolysis of ATP to ADP and P i , is very exergonic: ATP H11001 H 2 O 88n ADP H11001 P i H9004GH11032H11034 H11005 H1100230.5 kJ/mol These two reactions share the common intermediates P i and H 2 O and may be expressed as sequential reac- tions: (1) Glucose H11001 P i 88n glucose 6-phosphate H11001 H 2 O (2) ATP H11001 H 2 O 88n ADP H11001 P i Sum: ATP H11001 glucose 88n ADP H11001 glucose 6-phosphate The overall standard free-energy change is obtained by adding the H9004GH11032H11034 values for individual reactions: H9004GH11032H11034 H11005 13.8 kJ/mol H11001 (H1100230.5 kJ/mol) H11005H1100216.7 kJ/mol The overall reaction is exergonic. In this case, energy stored in ATP is used to drive the synthesis of glucose 6-phosphate, even though its formation from glucose and inorganic phosphate (P i ) is endergonic. The path- way of glucose 6-phosphate formation by phosphoryl transfer from ATP is different from reactions (1) and (2) above, but the net result is the same as the sum of the two reactions. In thermodynamic calculations, all that matters is the state of the system at the beginning of the process and its state at the end; the route be- tween the initial and final states is immaterial. We have said that H9004GH11032H11034 is a way of expressing the equilibrium constant for a reaction. For reaction (1) above, KH11032 eq 1 H11005H110053.9 H11003 10 H110023 M H110021 Notice that H 2 O is not included in this expression, as its concentration (55.5 M) is assumed to remain unchanged by the reaction. The equilibrium constant for the hy- drolysis of ATP is KH11032 eq 2 H11005 H5007 [A [ D A P T ] P [P ] i ] H5007 H11005 2.0 H11003 10 5 M The equilibrium constant for the two coupled reactions is KH11032 eq 3 H11005 H11005 (KH11032 eq 1 )(KH11032 eq 2 ) H11005 (3.9 H11003 10 H110023 M H110021 ) (2.0 H11003 10 5 M) H11005 7.8 H11003 10 2 This calculation illustrates an important point about equilibrium constants: although the H9004GH11032H11034 values for two reactions that sum to a third are additive, the KH11032 eq for a reaction that is the sum of two reactions is the prod- uct of their individual KH11032 eq values. Equilibrium constants are multiplicative. By coupling ATP hydrolysis to glu- [glucose 6-phosphate][ADP][P i ] H5007H5007H5007H5007 [glucose][P i ][ATP] [glucose 6-phosphate] H5007H5007H5007 [glucose][P i ] cose 6-phosphate synthesis, the KH11032 eq for formation of glucose 6-phosphate has been raised by a factor of about 2 H11003 10 5 . This common-intermediate strategy is employed by all living cells in the synthesis of metabolic intermediates and cellular components. Obviously, the strategy works only if compounds such as ATP are continuously avail- able. In the following chapters we consider several of the most important cellular pathways for producing ATP. SUMMARY 13.1 Bioenergetics and Thermodynamics ■ Living cells constantly perform work. They require energy for maintaining their highly organized structures, synthesizing cellular components, generating electric currents, and many other processes. ■ Bioenergetics is the quantitative study of energy relationships and energy conversions in biological systems. Biological energy transformations obey the laws of thermodynamics. ■ All chemical reactions are influenced by two forces: the tendency to achieve the most stable bonding state (for which enthalpy, H, is a useful expression) and the tendency to achieve the highest degree of randomness, expressed as entropy, S. The net driving force in a reaction is H9004G, the free-energy change, which represents the net effect of these two factors: H9004G H11005H9004H H11002 T H9004S. ■ The standard transformed free-energy change, H9004GH11032H11034, is a physical constant that is characteristic for a given reaction and can be calculated from the equilibrium constant for the reaction: H9004GH11032H11034 H11005 H11002RT ln KH11032 eq . ■ The actual free-energy change, H9004G, is a variable that depends on H9004GH11032H11034 and on the concentrations of reactants and products: H9004G H11005H9004GH11032H11034 H11001 RT ln ([products]/[reactants]). ■ When H9004G is large and negative, the reaction tends to go in the forward direction; when H9004G is large and positive, the reaction tends to go in the reverse direction; and when H9004G H11005 0, the system is at equilibrium. ■ The free-energy change for a reaction is independent of the pathway by which the reaction occurs. Free-energy changes are additive; the net chemical reaction that results from successive reactions sharing a common intermediate has an overall free-energy change that is the sum of the H9004G values for the individual reactions. 13.1 Bioenergetics and Thermodynamics 495 13.2 Phosphoryl Group Transfers and ATP Having developed some fundamental principles of en- ergy changes in chemical systems, we can now exam- ine the energy cycle in cells and the special role of ATP as the energy currency that links catabolism and an- abolism (see Fig. 1–28). Heterotrophic cells obtain free energy in a chemical form by the catabolism of nutrient molecules, and they use that energy to make ATP from ADP and P i . ATP then donates some of its chemical en- ergy to endergonic processes such as the synthesis of metabolic intermediates and macromolecules from smaller precursors, the transport of substances across membranes against concentration gradients, and me- chanical motion. This donation of energy from ATP gen- erally involves the covalent participation of ATP in the reaction that is to be driven, with the eventual result that ATP is converted to ADP and P i or, in some reac- tions, to AMP and 2 P i . We discuss here the chemical basis for the large free-energy changes that accompany hydrolysis of ATP and other high-energy phosphate compounds, and we show that most cases of energy donation by ATP involve group transfer, not simple hy- drolysis of ATP. To illustrate the range of energy trans- ductions in which ATP provides the energy, we consider the synthesis of information-rich macromolecules, the transport of solutes across membranes, and motion pro- duced by muscle contraction. The Free-Energy Change for ATP Hydrolysis Is Large and Negative Figure 13–1 summarizes the chemical basis for the rel- atively large, negative, standard free energy of hydrol- ysis of ATP. The hydrolytic cleavage of the terminal phosphoric acid anhydride (phosphoanhydride) bond in ATP separates one of the three negatively charged phosphates and thus relieves some of the electrostatic repulsion in ATP; the P i (HPO 4 2H11002 ) released is stabilized by the formation of several resonance forms not possi- ble in ATP; and ADP 2H11002 , the other direct product of hydrolysis, immediately ionizes, releasing H H11001 into a medium of very low [H H11001 ] (~10 H110027 M). Because the con- centrations of the direct products of ATP hydrolysis are, in the cell, far below the concentrations at equilibrium (Table 13–5), mass action favors the hydrolysis reaction in the cell. Although the hydrolysis of ATP is highly exergonic (H9004GH11032H11034 H11005 H1100230.5 kJ/mol), the molecule is kinetically sta- ble at pH 7 because the activation energy for ATP hydrolysis is relatively high. Rapid cleavage of the phos- phoanhydride bonds occurs only when catalyzed by an enzyme. The free-energy change for ATP hydrolysis is H1100230.5 kJ/mol under standard conditions, but the actual free energy of hydrolysis (H9004G) of ATP in living cells is very different: the cellular concentrations of ATP, ADP, and P i are not identical and are much lower than the 1.0 M of standard conditions (Table 13–5). Furthermore, Mg 2H11001 in the cytosol binds to ATP and ADP (Fig. 13–2), and for most enzymatic reactions that involve ATP as phosphoryl group donor, the true substrate is MgATP 2H11002 . The relevant H9004GH11032H11034 is therefore that for MgATP 2H11002 hy- drolysis. Box 13–1 shows how H9004G for ATP hydrolysis in the intact erythrocyte can be calculated from the data in Table 13–5. In intact cells, H9004G for ATP hydrolysis, usually designated H9004G p , is much more negative than Chapter 13 Principles of Bioenergetics496 ADP 3H11002 H11001 P i 2H11002 H11001 H H11001 H9004GH11032H11034 H11005 H1100230.5 kJ/mol ATP 4H11002 H11001 H 2 O A B PO P H11002 O O B A H11002 O O OO O O Rib AdenineO O OHO ADP 2H11546 A B H11002 O O O O OO Rib Adenine ADP 3H11546 POPO H11002 O O B A H11002 O O O H H11001 H11001 OP B A O H11002 O O PO H11002 O O B A H11002 O O O A O B P H11002 O O O O O OO Rib Adenine ATP 4H11546 H OH P i H11002 POO O A O O O POO O B A H11002 O O 3H11002 OH H9254 H11002 H9254 H11002 H9254 H11002 H9254 H11002 resonance stabilization A H H11001 2 ionization 3 hydrolysis, with relief of charge repulsion 1 FIGURE 13–1 Chemical basis for the large free-energy change asso- ciated with ATP hydrolysis. 1The charge separation that results from hydrolysis relieves electrostatic repulsion among the four negative charges on ATP. 2 The product inorganic phosphate (P i ) is stabilized by formation of a resonance hybrid, in which each of the four phos- phorus–oxygen bonds has the same degree of double-bond character and the hydrogen ion is not permanently associated with any one of the oxygens. (Some degree of resonance stabilization also occurs in phosphates involved in ester or anhydride linkages, but fewer reso- nance forms are possible than for P i .) 3 The product ADP 2H11002 imme- diately ionizes, releasing a proton into a medium of very low [H H11001 ] (pH 7). A fourth factor (not shown) that favors ATP hydrolysis is the greater degree of solvation (hydration) of the products P i and ADP rel- ative to ATP, which further stabilizes the products relative to the re- actants. H9004GH11032H11034, ranging from H1100250 to H1100265 kJ/mol. H9004G p is often called the phosphorylation potential. In the follow- ing discussions we use the standard free-energy change for ATP hydrolysis, because this allows comparison, on the same basis, with the energetics of other cellular reactions. Remember, however, that in living cells H9004G is the relevant quantity—for ATP hydrolysis and all other reactions—and may be quite different from H9004GH11032H11034. Other Phosphorylated Compounds and Thioesters Also Have Large Free Energies of Hydrolysis Phosphoenolpyruvate (Fig. 13–3) contains a phosphate ester bond that undergoes hydrolysis to yield the enol form of pyruvate, and this direct product can immedi- ately tautomerize to the more stable keto form of pyru- vate. Because the reactant (phosphoenolpyruvate) has only one form (enol) and the product (pyruvate) has two possible forms, the product is stabilized relative to the reactant. This is the greatest contributing factor to the high standard free energy of hydrolysis of phospho- enolpyruvate: H9004GH11032H11034 H11005 H1100261.9 kJ/mol. Another three-carbon compound, 1,3-bisphospho- glycerate (Fig. 13–4), contains an anhydride bond be- tween the carboxyl group at C-1 and phosphoric acid. Hydrolysis of this acyl phosphate is accompanied by a large, negative, standard free-energy change (H9004GH11032H11034 H11005 13.2 Phosphoryl Group Transfers and ATP 497 Concentration (mM)* ATP ADP ? AMP P i PCr Rat hepatocyte 3.38 1.32 0.29 4.8 0 Rat myocyte 8.05 0.93 0.04 8.05 28 Rat neuron 2.59 0.73 0.06 2.72 4.7 Human erythrocyte 2.25 0.25 0.02 1.65 0 E. coli cell 7.90 1.04 0.82 7.9 0 Adenine Nucleotide, Inorganic Phosphate, and Phosphocreatine Concentrations in Some Cells TABLE 13–5 *For erythrocytes the concentrations are those of the cytosol (human erythrocytes lack a nucleus and mitochondria). In the other types of cells the data are for the entire cell contents, although the cytosol and the mitochondria have very different concentrations of ADP. PCr is phosphocreatine, discussed on p. 489. ? This value reflects total concentration; the true value for free ADP may be much lower (see Box 13–1). OP Mg 2H11001 A H11002 OPO O B A O O O OO Rib Adenine MgADP H11546 OP B Mg 2H11001 A O H11002 O O PO H11002 O O B A H11002 O O O O B P H11002 O O O O O OO Rib Adenine MgATP 2H11546 ? ? ? ? O H11002 O H11002 O O B A FIGURE 13–2 Mg 2H11545 and ATP. Formation of Mg 2H11001 complexes partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP. O A tautomerization C J O G Pyruvate (keto form) H11002 O C CH 3 H 2 O P i B H11002 O P O GJ D C H9004GH11032H11034 H11005 H1100261.9 kJ/mol D PEP 3H11002 H11001 H 2 O J O G PEP G H11002 O pyruvate H11002 H11001 P i 2H11002 CH 2 O B OC D OH J O G Pyruvate (enol form) H11002 O C CH 2 O J hydrolysis O C O H11002 FIGURE 13–3 Hydrolysis of phosphoenol- pyruvate (PEP). Catalyzed by pyruvate kinase, this reaction is followed by spontaneous tautomerization of the product, pyruvate. Tautomerization is not possible in PEP, and thus the products of hydrolysis are stabilized relative to the reactants. Resonance stabilization of P i also occurs, as shown in Figure 13–1. H1100249.3 kJ/mol), which can, again, be explained in terms of the structure of reactant and products. When H 2 O is added across the anhydride bond of 1,3-bisphospho- glycerate, one of the direct products, 3-phosphoglyceric acid, can immediately lose a proton to give the car- boxylate ion, 3-phosphoglycerate, which has two equally probable resonance forms (Fig. 13–4). Removal of the direct product (3-phosphoglyceric acid) and formation of the resonance-stabilized ion favor the forward reaction. Chapter 13 Principles of Bioenergetics498 3-Phosphoglyceric acid hydrolysis A O M CHOH CH 2 D A A A A P O P O C O H11002 O OH O H11002 H H11001 H 2 O P i ionization 1,3-Bisphosphoglycerate 3-Phosphoglycerate D P O H11002 G H11002 O O H11002 O A O M O CHOH CH 2 D A A A A P O P O C O 3 1 2 J H11002 O G G resonance stabilization A O CHOH CH 2 D A A A A P O P O C O H11002 O O H11002 O H9254 H11002 H9254 H11002 H9004GH11032H11034 H11005 H1100249.3 kJ/mol 1,3-Bisphosphoglycerate 4H11002 H11001 H 2 O 3-phosphoglycerate 3H11002 H11001 P i 2H11002 H11001 H H11001 FIGURE 13–4 Hydrolysis of 1,3- bisphosphoglycerate. The direct product of hydrolysis is 3-phospho- glyceric acid, with an undissociated carboxylic acid group, but dissociation occurs immediately. This ionization and the resonance structures it makes possible stabilize the product relative to the reactants. Resonance stabilization of P i further contributes to the negative free- energy change. BOX 13–1 WORKING IN BIOCHEMISTRY The Free Energy of Hydrolysis of ATP within Cells: The Real Cost of Doing Metabolic Business The standard free energy of hydrolysis of ATP is H1100230.5 kJ/mol. In the cell, however, the concentrations of ATP, ADP, and P i are not only unequal but much lower than the standard 1 M concentrations (see Table 13–5). Moreover, the cellular pH may differ somewhat from the standard pH of 7.0. Thus the actual free energy of hydrolysis of ATP under intracellular con- ditions (H9004G p ) differs from the standard free-energy change, H9004GH11032H11034. We can easily calculate H9004G p . In human erythrocytes, for example, the concentra- tions of ATP, ADP, and P i are 2.25, 0.25, and 1.65 mM, respectively. Let us assume for simplicity that the pH is 7.0 and the temperature is 25 H11034C, the standard pH and temperature. The actual free energy of hydrolysis of ATP in the erythrocyte under these conditions is given by the relationship H9004G p H11005H9004GH11032H11034 H11001 RT lnH5007 [A [ D A P T ] P [P ] i ] H5007 Substituting the appropriate values we obtain H9004G p H11005H1100230.5 kJ/mol H11001 H20900 (8.315 J/mol H11080 K)(298 K) ln H20901 H11005H1100230.5 kJ/mol H11001 (2.48 kJ/mol) ln 1.8 H11003 10 H110024 H11005H1100230.5 kJ/mol H11002 21 kJ/mol H11005H1100252 kJ/mol Thus H9004G p , the actual free-energy change for ATP hy- drolysis in the intact erythrocyte (H1100252 kJ/mol), is much larger than the standard free-energy change (H1100230.5 kJ/mol). By the same token, the free energy required to synthesize ATP from ADP and P i under the conditions prevailing in the erythrocyte would be 52 kJ/mol. Because the concentrations of ATP, ADP, and P i differ from one cell type to another (see Table 13–5), H9004G p for ATP hydrolysis likewise differs among cells. Moreover, in any given cell, H9004G p can vary from time to time, depending on the metabolic conditions in the cell and how they influence the concentrations of ATP, ADP, P i , and H H11001 (pH). We can calculate the actual free-energy change for any given metabolic reaction as it occurs in the cell, providing we know the con- centrations of all the reactants and products of the re- action and know about the other factors (such as pH, temperature, and concentration of Mg 2H11001 ) that may af- fect the H9004GH11032H11034 and thus the calculated free-energy change, H9004G p . To further complicate the issue, the total concen- trations of ATP, ADP, P i , and H H11001 may be substantially higher than the free concentrations, which are the thermodynamically relevant values. The difference is due to tight binding of ATP, ADP, and P i to cellular proteins. For example, the concentration of free ADP in resting muscle has been variously estimated at be- tween 1 and 37 H9262M. Using the value 25 H9262M in the cal- culation outlined above, we get a H9004G p of H1100258 kJ/mol. Calculation of the exact value of H9004G p is perhaps less instructive than the generalization we can make about actual free-energy changes: in vivo, the energy released by ATP hydrolysis is greater than the stan- dard free-energy change, H9004GH11032H11034. (0.25 H11003 10 H110023 )(1.65 H11003 10 H110023 ) H5007H5007H5007H5007 2.25 H11003 10 H110023 In phosphocreatine (Fig. 13–5), the PON bond can be hydrolyzed to generate free creatine and P i . The re- lease of P i and the resonance stabilization of creatine favor the forward reaction. The standard free-energy change of phosphocreatine hydrolysis is again large, H1100243.0 kJ/mol. In all these phosphate-releasing reactions, the sev- eral resonance forms available to P i (Fig. 13–1) stabi- lize this product relative to the reactant, contributing to an already negative free-energy change. Table 13–6 lists the standard free energies of hydrolysis for a number of phosphorylated compounds. Thioesters, in which a sulfur atom replaces the usual oxygen in the ester bond, also have large, nega- tive, standard free energies of hydrolysis. Acetyl-coen- zyme A, or acetyl-CoA (Fig. 13–6), is one of many thioesters important in metabolism. The acyl group in these compounds is activated for transacylation, con- densation, or oxidation-reduction reactions. Thioesters undergo much less resonance stabilization than do oxy- gen esters; consequently, the difference in free energy between the reactant and its hydrolysis products, which are resonance-stabilized, is greater for thioesters than for comparable oxygen esters (Fig. 13–7). In both cases, hydrolysis of the ester generates a carboxylic acid, which can ionize and assume several resonance forms. Together, these factors result in the large, negative H9004GH11032H11034 (H1100231 kJ/mol) for acetyl-CoA hydrolysis. To summarize, for hydrolysis reactions with large, negative, standard free-energy changes, the products are more stable than the reactants for one or more of the following reasons: (1) the bond strain in reactants due to electrostatic repulsion is relieved by charge sep- aration, as for ATP; (2) the products are stabilized by 13.2 Phosphoryl Group Transfers and ATP 499 H11001 NH 2 COO H11002 resonance stabilization P i hydrolysis A B H11002 O OOO O A CH 2 N H 2 O O B H11001 NH 2 COO H11002 O CH 3 A P B OOO A A CH 2 N H C Creatine H 2 N H 2 N CN COO H11002 A A CH 2 CH 3 CH 3 C O N O H11002 H9004GH11032H11034 H11005 H1100243.0 kJ/mol creatine H11001 P i 2H11002 Phosphocreatine 2H11002 H11001 H 2 O Phosphocreatine H 2 N H9254 H11001 H9254 H11001 H9254 H11001 FIGURE 13–5 Hydrolysis of phospho- creatine. Breakage of the PON bond in phosphocreatine produces creatine, which is stabilized by formation of a resonance hybrid. The other product, P i , is also resonance stabilized. H9004GH11032H11034 (kJ/mol) (kcal/mol) Phosphoenolpyruvate H1100261.9 H1100214.8 1,3-bisphosphoglycerate (n 3-phosphoglycerate H11001 P i ) H1100249.3 H1100211.8 Phosphocreatine H1100243.0 H1100210.3 ADP (n AMP H11001 P i ) H1100232.8 H110027.8 ATP (n ADP H11001 P i ) H1100230.5 H110027.3 ATP (n AMP H11001 PP i ) H1100245.6 H1100210.9 AMP (n adenosine H11001 P i ) H1100214.2 H110023.4 PP i (n 2P i ) H1100219.2 H110024.0 Glucose 1-phosphate H1100220.9 H110025.0 Fructose 6-phosphate H1100215.9 H110023.8 Glucose 6-phosphate H1100213.8 H110023.3 Glycerol 1-phosphate H110029.2 H110022.2 Acetyl-CoA H1100231.4 H110027.5 Standard Free Energies of Hydrolysis of Some Phosphorylated Compounds and Acetyl-CoA (a Thioester) TABLE 13–6 Source: Data mostly from Jencks, W.P. (1976) in Handbook of Biochemistry and Molecular Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. I, pp. 296–304, CRC Press, Boca Raton, FL. The value for the free energy of hydrolysis of PP i is from Frey, P.A. & Arabshahi, A. (1995) Standard free-energy change for the hydrolysis of the H9251–H9252- phosphoanhydride bridge in ATP. Biochemistry 34, 11,307–11,310. CH 3 H9004GH11032H11034 H11005 H1100231.4 kJ/mol acetate H11002 H11001 CoA C O OH Acetate O Acetyl- H 2 O resonance stabilization CoASH hydrolysis ionization G J S-CoA CH 3 C O O G J CH 3 C O O Acetyl-CoA Acetic acid D G H H11001 O CoA H11001 H 2 O H11001 H H11001 H9254 H11002 H9254 H11002 FIGURE 13–6 Hydrolysis of acetyl-coenzyme A. Acetyl-CoA is a thioester with a large, negative, standard free energy of hydrolysis. Thioesters contain a sulfur atom in the position occupied by an oxy- gen atom in oxygen esters. The complete structure of coenzyme A (CoA, or CoASH) is shown in Figure 8–41. ionization, as for ATP, acyl phosphates, and thioesters; (3) the products are stabilized by isomerization (tau- tomerization), as for phosphoenolpyruvate; and/or (4) the products are stabilized by resonance, as for creatine released from phosphocreatine, carboxylate ion re- leased from acyl phosphates and thioesters, and phos- phate (P i ) released from anhydride or ester linkages. ATP Provides Energy by Group Transfers, Not by Simple Hydrolysis Throughout this book you will encounter reactions or processes for which ATP supplies energy, and the con- tribution of ATP to these reactions is commonly indi- cated as in Figure 13–8a, with a single arrow showing the conversion of ATP to ADP and P i (or, in some cases, of ATP to AMP and pyrophosphate, PP i ). When written this way, these reactions of ATP appear to be simple hy- drolysis reactions in which water displaces P i (or PP i ), and one is tempted to say that an ATP-dependent re- action is “driven by the hydrolysis of ATP.” This is not the case. ATP hydrolysis per se usually accomplishes nothing but the liberation of heat, which cannot drive a chemical process in an isothermal system. A single re- action arrow such as that in Figure 13–8a almost in- variably represents a two-step process (Fig. 13–8b) in which part of the ATP molecule, a phosphoryl or py- rophosphoryl group or the adenylate moiety (AMP), is first transferred to a substrate molecule or to an amino acid residue in an enzyme, becoming covalently at- tached to the substrate or the enzyme and raising its free-energy content. Then, in a second step, the phos- phate-containing moiety transferred in the first step is displaced, generating P i , PP i , or AMP. Thus ATP partic- ipates covalently in the enzyme-catalyzed reaction to which it contributes free energy. Some processes do involve direct hydrolysis of ATP (or GTP), however. For example, noncovalent binding of ATP (or of GTP), followed by its hydrolysis to ADP (or GDP) and P i , can provide the energy to cycle some proteins between two conformations, producing me- chanical motion. This occurs in muscle contraction and in the movement of enzymes along DNA or of ribosomes along messenger RNA. The energy-dependent reactions catalyzed by helicases, RecA protein, and some topo- isomerases (Chapter 25) also involve direct hydrolysis of phosphoanhydride bonds. GTP-binding proteins that act in signaling pathways directly hydrolyze GTP to drive conformational changes that terminate signals Chapter 13 Principles of Bioenergetics500 H11001 CH 2 NH 2 CHH 3 N ATP ADP C O A O GJ A A A CH 2 COO H11002 H11001 CH 2 H 3 N O G J A A NH 3 Glutamate H11001 H11001 CH 2 CHH 3 N C O A O GJ A A A CH 2 COO H11002 NH 3 G C O J O O H11002 G O H11002 ATP ADP H11001 P i CH A O A CH 2 COO H11002 D P glutamyl phosphate H11002 O Enzyme-bound P i Glutamine 21 (a) Written as a one-step reaction (b) Actual two-step reaction FIGURE 13–8 ATP hydrolysis in two steps. (a) The contribution of ATP to a reaction is often shown as a single step, but is almost always a two-step process. (b) Shown here is the reaction catalyzed by ATP- dependent glutamine synthetase. 1 A phosphoryl group is transferred from ATP to glutamate, then 2 the phosphoryl group is displaced by NH 3 and released as P i . O CH 3 C CH 3 O C O Thioester J O O R OH Extra stabilization of oxygen ester by resonance CH 3 C G J O O O OR H11001 R OH CH 3 C G J O O S OSH F ree energy , G resonance stabilization Oxygen ester CH 3 C G J O O OH H11001 R OR O G H9004G for oxygen ester hydrolysis H9004G for thioester hydrolysis H9254 H11002 H9254 H11002 FIGURE 13–7 Free energy of hydrolysis for thioesters and oxygen esters. The products of both types of hydrolysis reaction have about the same free-energy content (G), but the thioester has a higher free-energy content than the oxygen ester. Orbital overlap between the O and C atoms allows resonance stabilization in oxygen esters; orbital overlap between S and C atoms is poorer and provides little resonance stabilization. triggered by hormones or by other extracellular factors (Chapter 12). The phosphate compounds found in living organisms can be divided somewhat arbitrarily into two groups, based on their standard free energies of hydrolysis (Fig. 13–9). “High-energy” compounds have a H9004GH11032H11034 of hydrolysis more negative than H1100225 kJ/mol; “low-energy” compounds have a less negative H9004GH11032H11034. Based on this cri- terion, ATP, with a H9004GH11032H11034 of hydrolysis of H1100230.5 kJ/mol (H110027.3 kcal/mol), is a high-energy compound; glucose 6-phosphate, with a H9004GH11032H11034 of hydrolysis of H1100213.8 kJ/mol (H110023.3 kcal/mol), is a low-energy compound. The term “high-energy phosphate bond,” long used by biochemists to describe the POO bond broken in hy- drolysis reactions, is incorrect and misleading as it wrongly suggests that the bond itself contains the en- ergy. In fact, the breaking of all chemical bonds requires an input of energy. The free energy released by hy- drolysis of phosphate compounds does not come from the specific bond that is broken; it results from the prod- ucts of the reaction having a lower free-energy content than the reactants. For simplicity, we will sometimes use the term “high-energy phosphate compound” when re- ferring to ATP or other phosphate compounds with a large, negative, standard free energy of hydrolysis. As is evident from the additivity of free-energy changes of sequential reactions, any phosphorylated compound can be synthesized by coupling the synthe- sis to the breakdown of another phosphorylated com- pound with a more negative free energy of hydrolysis. For example, because cleavage of P i from phospho- enolpyruvate (PEP) releases more energy than is needed to drive the condensation of P i with ADP, the direct donation of a phosphoryl group from PEP to ADP is thermodynamically feasible: H9004GH11032H11034 (kJ/mol) (1) PEP H11001 H 2 O 8n pyruvate H11001 P i H1100261.9 (2) ADP H11001 P i 8n ATP H11001 H 2 O H1100130.5 Sum: PEP H11001 ADP 8n pyruvate H11001 ATP H1100231.4 Notice that while the overall reaction above is repre- sented as the algebraic sum of the first two reactions, the overall reaction is actually a third, distinct reaction that does not involve P i ; PEP donates a phosphoryl group directly to ADP. We can describe phosphorylated compounds as having a high or low phosphoryl group transfer potential, on the basis of their standard free en- ergies of hydrolysis (as listed in Table 13–6). The phos- phoryl group transfer potential of phosphoenolpyruvate is very high, that of ATP is high, and that of glucose 6- phosphate is low (Fig. 13–9). Much of catabolism is directed toward the synthesis of high-energy phosphate compounds, but their forma- tion is not an end in itself; they are the means of acti- vating a very wide variety of compounds for further chemical transformation. The transfer of a phosphoryl group to a compound effectively puts free energy into that compound, so that it has more free energy to give up during subsequent metabolic transformations. We de- scribed above how the synthesis of glucose 6-phosphate is accomplished by phosphoryl group transfer from ATP. In the next chapter we see how this phosphorylation of glucose activates, or “primes,” the glucose for catabolic reactions that occur in nearly every living cell. Because of its intermediate position on the scale of group trans- fer potential, ATP can carry energy from high-energy 13.2 Phosphoryl Group Transfers and ATP 501 H9004 G H11032H11034 of hydrolysis (kJ/mol) P i P O O A CHOH D H1100210 C M CH 2 A O Creatine Phosphoenolpyruvate H1100270 OP 1,3-Bisphosphoglycerate PORib Glycerol- OO P P H1100260 H1100230 H1100250 H1100240 H1100220 P ATP Low-energy compounds PGlucose 6- High-energy compounds Adenine COO H11002 B C A CH 2 O P P Phosphocreatine O OOO OO 0 FIGURE 13–9 Ranking of biological phosphate compounds by standard free energies of hydrol- ysis. This shows the flow of phosphoryl groups, represented by P , from high-energy phosphoryl donors via ATP to acceptor molecules (such as glucose and glycerol) to form their low-energy phosphate derivatives. This flow of phosphoryl groups, catalyzed by enzymes called kinases, proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of low- energy phosphate compounds releases P i , which has an even lower phosphoryl group transfer potential (as defined in the text). phosphate compounds produced by catabolism to com- pounds such as glucose, converting them into more re- active species. ATP thus serves as the universal energy currency in all living cells. One more chemical feature of ATP is crucial to its role in metabolism: although in aqueous solution ATP is thermodynamically unstable and is therefore a good phosphoryl group donor, it is kinetically stable. Because of the huge activation energies (200 to 400 kJ/mol) re- quired for uncatalyzed cleavage of its phosphoanhydride bonds, ATP does not spontaneously donate phosphoryl groups to water or to the hundreds of other potential acceptors in the cell. Only when specific enzymes are present to lower the energy of activation does phos- phoryl group transfer from ATP proceed. The cell is therefore able to regulate the disposition of the energy carried by ATP by regulating the various enzymes that act on it. ATP Donates Phosphoryl, Pyrophosphoryl, and Adenylyl Groups The reactions of ATP are generally S N 2 nucleophilic dis- placements (p. II.8), in which the nucleophile may be, for example, the oxygen of an alcohol or carboxylate, or a nitrogen of creatine or of the side chain of arginine or histidine. Each of the three phosphates of ATP is sus- ceptible to nucleophilic attack (Fig. 13–10), and each position of attack yields a different type of product. Nucleophilic attack by an alcohol on the H9253 phos- phate (Fig. 13–10a) displaces ADP and produces a new phosphate ester. Studies with 18 O-labeled reactants have shown that the bridge oxygen in the new com- pound is derived from the alcohol, not from ATP; the group transferred from ATP is a phosphoryl (OPO 3 2H11002 ), not a phosphate (OOPO 3 2H11002 ). Phosphoryl group transfer from ATP to glutamate (Fig. 13–8) or to glucose (p. 218) involves attack at the H9253 position of the ATP molecule. Attack at the H9252 phosphate of ATP displaces AMP and transfers a pyrophosphoryl (not pyrophosphate) group to the attacking nucleophile (Fig. 13–10b). For exam- ple, the formation of 5H11032-phosphoribosyl-1-pyrophosphate (p. XXX), a key intermediate in nucleotide synthesis, results from attack of an OOH of the ribose on the H9252 phosphate. Nucleophilic attack at the H9251 position of ATP displaces PP i and transfers adenylate (5H11032-AMP) as an adenylyl group (Fig. 13–10c); the reaction is an adenylylation (a-denH11032-i-li-la - H11032-shun, probably the most ungainly word in the biochemical language). Notice that hydrolysis of the H9251–H9252 phosphoanhydride bond releases considerably more energy (~46 kJ/mol) than hydrolysis of the H9252–H9253 bond (~31 kJ/mol) (Table 13–6). Furthermore, the PP i formed as a byproduct of the adenylylation is hydrolyzed to two P i by the ubiquitous enzyme inorganic pyro- phosphatase, releasing 19 kJ/mol and thereby provid- ing a further energy “push” for the adenylylation reac- tion. In effect, both phosphoanhydride bonds of ATP are split in the overall reaction. Adenylylation reactions are therefore thermodynamically very favorable. When the energy of ATP is used to drive a particularly unfavor- able metabolic reaction, adenylylation is often the mech- anism of energy coupling. Fatty acid activation is a good example of this energy-coupling strategy. The first step in the activation of a fatty acid— either for energy-yielding oxidation or for use in the syn- thesis of more complex lipids—is the formation of its thiol ester (see Fig. 17–5). The direct condensation of a fatty acid with coenzyme A is endergonic, but the for- mation of fatty acyl–CoA is made exergonic by stepwise removal of two phosphoryl groups from ATP. First, adenylate (AMP) is transferred from ATP to the car- boxyl group of the fatty acid, forming a mixed anhydride Chapter 13 Principles of Bioenergetics502 O OP O H11002 Rib Adenine H11002 O Pyrophosphoryl transfer (b) Phosphoryl transfer (a) Adenylyl transfer (c) Rib Adenine H9253 H9252H9251 R 18 OR 18 OR 18 O R 18 O R 18 O R 18 OR 18 O H11001 H11001 H11001 ADP AMP PP i O OP O H11002 O OP O H11002 O O H11002 P O H11002 O OP O H11002 O OP O H11002 O O H11002 P O H11002 Three positions on ATP for attack by the nucleophile FIGURE 13–10 Nucleophilic displacement reac- tions of ATP. Any of the three P atoms (H9251, H9252, or H9253) may serve as the electrophilic target for nucleophilic attack—in this case, by the labeled nucleophile RO 18 O:. The nucleophile may be an alcohol (ROH), a carboxyl group (RCOO H11002 ), or a phosphoanhydride (a nucleoside mono- or diphosphate, for example). (a) When the oxygen of the nucleophile attacks the H9253 position, the bridge oxygen of the product is labeled, indicating that the group transferred from ATP is a phosphoryl (OPO 3 2H11002 ), not a phosphate (OOPO 3 2H11002 ). (b) Attack on the H9252 position displaces AMP and leads to the transfer of a pyrophosphoryl (not pyrophosphate) group to the nucleophile. (c) Attack on the H9251 position displaces PP i and transfers the adenylyl group to the nucleophile. (fatty acyl adenylate) and liberating PP i . The thiol group of coenzyme A then displaces the adenylate group and forms a thioester with the fatty acid. The sum of these two reactions is energetically equivalent to the exer- gonic hydrolysis of ATP to AMP and PP i (H9004GH11032H11034 H11005 H1100245.6 kJ/mol) and the endergonic formation of fatty acyl–CoA (H9004GH11032H11034 H11005 31.4 kJ/mol). The formation of fatty acyl–CoA is made energetically favorable by hydrolysis of the PP i by inorganic pyrophosphatase. Thus, in the activation of a fatty acid, both phosphoanhydride bonds of ATP are broken. The resulting H9004GH11032H11034 is the sum of the H9004GH11032H11034 values for the breakage of these bonds, or H1100245.6 kJ/mol H11001 (H1100219.2) kJ/mol: ATP H11001 2H 2 O 88n AMP H11001 2Pi H9004GH11032H11034 H11005 H1100264.8 kJ/mol The activation of amino acids before their polymer- ization into proteins (see Fig. 27–14) is accomplished by an analogous set of reactions in which a transfer RNA molecule takes the place of coenzyme A. An interesting use of the cleavage of ATP to AMP and PP i occurs in the firefly, which uses ATP as an energy source to pro- duce light flashes (Box 13–2). 13.2 Phosphoryl Group Transfers and ATP 503 BOX 13–2 THE WORLD OF BIOCHEMISTRY Firefly Flashes: Glowing Reports of ATP Bioluminescence requires considerable amounts of energy. In the firefly, ATP is used in a set of reactions that converts chemical energy into light energy. In the 1950s, from many thousands of fireflies collected by children in and around Baltimore, William McElroy and his colleagues at The Johns Hopkins University isolated the principal biochemical components: lu- ciferin, a complex carboxylic acid, and luciferase, an enzyme. The generation of a light flash requires acti- vation of luciferin by an enzymatic reaction involving pyrophosphate cleavage of ATP to form luciferyl adenylate. In the presence of molecular oxygen and luciferase, the luciferin undergoes a multistep oxida- tive decarboxylation to oxyluciferin. This process is accompanied by emission of light. The color of the light flash differs with the firefly species and appears to be determined by differences in the structure of the luciferase. Luciferin is regenerated from oxyluciferin in a subsequent series of reactions. In the laboratory, pure firefly luciferin and lu- ciferase are used to measure minute quantities of ATP by the intensity of the light flash produced. As little as a few picomoles (10 H1100212 mol) of ATP can be meas- ured in this way. An enlightening extension of the studies in luciferase was the cloning of the luciferase gene into tobacco plants. When watered with a solu- tion containing luciferin, the plants glowed in the dark (see Fig. 9–29). A S POO O H11002 N HO S C Oxyluciferin regenerating reactions CO 2 H11001 AMP luciferase light O 2 AMP N H H ATP S N HO S COO H11002 N Adenine O Rib PP i Luciferin Luciferyl adenylate O H H H H S N HO S N O The firefly, a beetle of the Lampyridae family. Important components in the firefly bioluminescence cycle. Assembly of Informational Macromolecules Requires Energy When simple precursors are assembled into high mo- lecular weight polymers with defined sequences (DNA, RNA, proteins), as described in detail in Part III, energy is required both for the condensation of monomeric units and for the creation of ordered sequences. The precursors for DNA and RNA synthesis are nucleoside triphosphates, and polymerization is accompanied by cleavage of the phosphoanhydride linkage between the H9251 and H9252 phosphates, with the release of PP i (Fig. 13–11). The moieties transferred to the growing polymer in these reactions are adenylate (AMP), guanylate (GMP), cytidylate (CMP), or uridylate (UMP) for RNA synthe- sis, and their deoxy analogs (with TMP in place of UMP) for DNA synthesis. As noted above, the activation of amino acids for protein synthesis involves the donation of adenylate groups from ATP, and we shall see in Chap- ter 27 that several steps of protein synthesis on the ri- bosome are also accompanied by GTP hydrolysis. In all these cases, the exergonic breakdown of a nucleoside triphosphate is coupled to the endergonic process of synthesizing a polymer of a specific sequence. ATP Energizes Active Transport and Muscle Contraction ATP can supply the energy for transporting an ion or a molecule across a membrane into another aqueous com- partment where its concentration is higher (see Fig. 11–36). Transport processes are major consumers of en- ergy; in human kidney and brain, for example, as much as two-thirds of the energy consumed at rest is used to pump Na H11001 and K H11001 across plasma membranes via the Na H11001 K H11001 ATPase. The transport of Na H11001 and K H11001 is driven by cyclic phosphorylation and dephosphorylation of the transporter protein, with ATP as the phosphoryl group donor (see Fig. 11–37). Na H11001 -dependent phosphorylation of the Na H11001 K H11001 ATPase forces a change in the protein’s conformation, and K H11001 -dependent dephosphorylation favors return to the original conformation. Each cycle in the transport process results in the conversion of ATP to ADP and P i , and it is the free-energy change of ATP hydrolysis that drives the cyclic changes in protein con- formation that result in the electrogenic pumping of Na H11001 and K H11001 . Note that in this case ATP interacts covalently by phosphoryl group transfer to the enzyme, not the substrate. In the contractile system of skeletal muscle cells, myosin and actin are specialized to transduce the chem- ical energy of ATP into motion (see Fig. 5–33). ATP binds tightly but noncovalently to one conformation of myosin, holding the protein in that conformation. When myosin catalyzes the hydrolysis of its bound ATP, the ADP and P i dissociate from the protein, allowing it to relax into a second conformation until another molecule of ATP binds. The binding and subsequent hydrolysis of ATP (by myosin ATPase) provide the energy that forces cyclic changes in the conformation of the myosin head. The change in conformation of many individual myosin Chapter 13 Principles of Bioenergetics504 GTP CH 2 P A A H11002 O OH H O HH H OH O Guanine O P A O A O H11002 O A O O O POP P OO H11002 A OH H HH H O BaseCH 2 RNA chain lengthened by one nucleotide P A O A O H11002 O A O O OH H HH H OH O BaseCH 2 O P A OO O OOO O H11002 BB A O H11002 P P P O 2P i A OH H O HH H OH O Guanine A RNA chain A H11002 O O H11002 P O P A OO O OOO BB A O H11002 H11002 O O H11002 OCH 2 PP i first anhydride bond broken second anhydride bond broken S H9251H9252H9253 FIGURE 13–11 Nucleoside triphosphates in RNA synthesis. With each nucleoside monophosphate added to the growing chain, one PP i is released and hydrolyzed to two P i . The hydrolysis of two phospho- anhydride bonds for each nucleotide added provides the energy for forming the bonds in the RNA polymer and for assembling a specific sequence of nucleotides. molecules results in the sliding of myosin fibrils along actin filaments (see Fig. 5–32), which translates into macroscopic contraction of the muscle fiber. As we noted earlier, this production of mechanical motion at the expense of ATP is one of the few cases in which ATP hydrolysis per se, rather than group trans- fer from ATP, is the source of the chemical energy in a coupled process. Transphosphorylations between Nucleotides Occur in All Cell Types Although we have focused on ATP as the cell’s energy currency and donor of phosphoryl groups, all other nu- cleoside triphosphates (GTP, UTP, and CTP) and all the deoxynucleoside triphosphates (dATP, dGTP, dTTP, and dCTP) are energetically equivalent to ATP. The free- energy changes associated with hydrolysis of their phosphoanhydride linkages are very nearly identical with those shown in Table 13–6 for ATP. In preparation for their various biological roles, these other nucleotides are generated and maintained as the nucleoside triphos- phate (NTP) forms by phosphoryl group transfer to the corresponding nucleoside diphosphates (NDPs) and monophosphates (NMPs). ATP is the primary high-energy phosphate com- pound produced by catabolism, in the processes of gly- colysis, oxidative phosphorylation, and, in photosyn- thetic cells, photophosphorylation. Several enzymes then carry phosphoryl groups from ATP to the other nu- cleotides. Nucleoside diphosphate kinase, found in all cells, catalyzes the reaction ATP H11001 NDP (or dNDP) ADP H11001 NTP (or dNTP) DGH11032H11034 H11015 0 Although this reaction is fully reversible, the relatively high [ATP]/[ADP] ratio in cells normally drives the re- action to the right, with the net formation of NTPs and dNTPs. The enzyme actually catalyzes a two-step phos- phoryl transfer, which is a classic case of a double-dis- placement (Ping-Pong) mechanism (Fig. 13–12; see also Fig. 6–13b). First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme Mg 2H11001 3:::4 intermediate; then the phosphoryl group is transferred from the P –His residue to an NDP acceptor. Because the enzyme is nonspecific for the base in the NDP and works equally well on dNDPs and NDPs, it can synthe- size all NTPs and dNTPs, given the corresponding NDPs and a supply of ATP. Phosphoryl group transfers from ATP result in an accumulation of ADP; for example, when muscle is con- tracting vigorously, ADP accumulates and interferes with ATP-dependent contraction. During periods of in- tense demand for ATP, the cell lowers the ADP con- centration, and at the same time acquires ATP, by the action of adenylate kinase: 2ADP ATP H11001 AMP DGH11032H11034 H11015 0 This reaction is fully reversible, so after the intense de- mand for ATP ends, the enzyme can recycle AMP by converting it to ADP, which can then be phosphorylated to ATP in mitochondria. A similar enzyme, guanylate ki- nase, converts GMP to GDP at the expense of ATP. By pathways such as these, energy conserved in the cata- bolic production of ATP is used to supply the cell with all required NTPs and dNTPs. Phosphocreatine (Fig. 13–5), also called creatine phosphate, serves as a ready source of phosphoryl groups for the quick synthesis of ATP from ADP. The phosphocreatine (PCr) concentration in skeletal mus- cle is approximately 30 mM, nearly ten times the con- centration of ATP, and in other tissues such as smooth muscle, brain, and kidney [PCr] is 5 to 10 mM. The en- zyme creatine kinase catalyzes the reversible reaction ADP H11001 PCr ATP H11001 Cr DGH11032H11034 H11005 H1100212.5 kJ/mol When a sudden demand for energy depletes ATP, the PCr reservoir is used to replenish ATP at a rate consid- erably faster than ATP can be synthesized by catabolic pathways. When the demand for energy slackens, ATP produced by catabolism is used to replenish the PCr reservoir by reversal of the creatine kinase reaction. Or- ganisms in the lower phyla employ other PCr-like mole- cules (collectively called phosphagens) as phosphoryl reservoirs. Mg 2H11001 3:::4 Mg 2H11001 3:::4 13.2 Phosphoryl Group Transfers and ATP 505 PP PAdenosine (ATP) P PAdenosine (ADP) PP P P Enz His Enz His Ping Pong Nucleoside (any NTP or dNTP) P PNucleoside (any NDP or dNDP) FIGURE 13–12 Ping-Pong mechanism of nucleoside diphosphate kinase. The enzyme binds its first substrate (ATP in our example), and a phosphoryl group is transferred to the side chain of a His residue. ADP departs, and another nucleoside (or deoxynucleoside) diphos- phate replaces it, and this is converted to the corresponding triphos- phate by transfer of the phosphoryl group from the phosphohistidine residue. Inorganic Polyphosphate Is a Potential Phosphoryl Group Donor Inorganic polyphosphate (polyP) is a linear polymer composed of many tens or hundreds of P i residues linked through phosphoanhydride bonds. This polymer, present in all organisms, may accumulate to high levels in some cells. In yeast, for example, the amount of polyP that accumulates in the vacuoles would represent, if dis- tributed uniformly throughout the cell, a concentration of 200 mM! (Compare this with the concentrations of other phosphoryl donors listed in Table 13–5.) One potential role for polyP is to serve as a phos- phagen, a reservoir of phosphoryl groups that can be used to generate ATP, as creatine phosphate is used in muscle. PolyP has about the same phosphoryl group transfer potential as PP i . The shortest polyphosphate, PP i (n H11005 2), can serve as the energy source for active transport of H H11001 in plant vacuoles. For at least one form of the enzyme phosphofructokinase in plants, PP i is the phosphoryl group donor, a role played by ATP in ani- mals and microbes (p. XXX). The finding of high con- centrations of polyP in volcanic condensates and steam vents suggests that it could have served as an energy source in prebiotic and early cellular evolution. In prokaryotes, the enzyme polyphosphate ki- nase-1 (PPK-1) catalyzes the reversible reaction ATP H11001 polyP n ADP H11001 polyP nH110011 DGH11032H11034 H11005 H1100220 kJ/mol by a mechanism involving an enzyme-bound phospho- histidine intermediate (recall the mechanism of nucle- oside diphosphate kinase, described above). A second enzyme, polyphosphate kinase-2 (PPK-2), catalyzes the reversible synthesis of GTP (or ATP) from poly- phosphate and GDP (or ADP): GDP H11001 polyP nH110011 GTP H11001 polyP n PPK-2 is believed to act primarily in the direction of GTP and ATP synthesis, and PPK-1 in the direction of polyphosphate synthesis. PPK-1 and PPK-2 are present in a wide variety of prokaryotes, including many patho- genic bacteria. In prokaryotes, elevated levels of polyP have been shown to promote expression of a number of genes in- volved in adaptation of the organism to conditions of starvation or other threats to survival. In Escherichia coli, for example, polyP accumulates when cells are starved for amino acids or P i , and this accumulation con- Mn 2H11001 3:::4 Mg 2H11001 3:::4 O OP O H11002 O OP O H11002 O OP O H11002 O OP O H11002 O OP O H11002 H11002 O Inorganic polyphosphate (polyP) fers a survival advantage. Deletion of the genes for polyphosphate kinases diminishes the ability of certain pathogenic bacteria to invade animal tissues. The en- zymes may therefore prove to be vulnerable targets in the development of new antimicrobial drugs. No gene in yeast encodes a PPK-like protein, but four genes—unrelated to bacterial PPK genes—are nec- essary for the synthesis of polyphosphate. The mecha- nism for polyphosphate synthesis in eukaryotes seems to be quite different from that in prokaryotes. Biochemical and Chemical Equations Are Not Identical Biochemists write metabolic equations in a simplified way, and this is particularly evident for reactions in- volving ATP. Phosphorylated compounds can exist in several ionization states and, as we have noted, the dif- ferent species can bind Mg 2H11001 . For example, at pH 7 and 2mM Mg 2H11001 , ATP exists in the forms ATP 4H11002 , HATP 3H11002 , H 2 ATP 2H11002 , MgHATP H11002 , and Mg 2 ATP. In thinking about the biological role of ATP, however, we are not always in- terested in all this detail, and so we consider ATP as an entity made up of a sum of species, and we write its hy- drolysis as the biochemical equation ATP H11001 H 2 O 8n ADP H11001 P i where ATP, ADP, and P i are sums of species. The corresponding apparent equilibrium constant, KH11032 eq H11005 [ADP][P i ]/[ATP], depends on the pH and the concentra- tion of free Mg 2H11001 . Note that H H11001 and Mg 2H11001 do not ap- pear in the biochemical equation because they are held constant. Thus a biochemical equation does not balance H, Mg, or charge, although it does balance all other el- ements involved in the reaction (C, N, O, and P in the equation above). We can write a chemical equation that does balance for all elements and for charge. For example, when ATP is hydrolyzed at a pH above 8.5 in the absence of Mg 2H11001 , the chemical reaction is represented by ATP 4H11002 H11001 H 2 O 8n ADP 3H11002 H11001 HPO 4 2H11002 H11001 H H11001 The corresponding equilibrium constant, KH11032 eq H11005 [ADP 3H11002 ][HPO 4 2H11002 ][H H11001 ]/[ATP 4H11002 ], depends only on tem- perature, pressure, and ionic strength. Both ways of writing a metabolic reaction have value in biochemistry. Chemical equations are needed when we want to account for all atoms and charges in a re- action, as when we are considering the mechanism of a chemical reaction. Biochemical equations are used to determine in which direction a reaction will proceed spontaneously, given a specified pH and [Mg 2H11001 ], or to calculate the equilibrium constant of such a reaction. Throughout this book we use biochemical equa- tions, unless the focus is on chemical mechanism, and we use values of H9004GH11032H11034 and KH11032 eq as determined at pH 7 and 1 mM Mg 2H11001 . Chapter 13 Principles of Bioenergetics506 SUMMARY 13.2 Phosphoryl Group Transfers and ATP ■ ATP is the chemical link between catabolism and anabolism. It is the energy currency of the living cell. The exergonic conversion of ATP to ADP and P i , or to AMP and PP i , is coupled to many endergonic reactions and processes. ■ Direct hydrolysis of ATP is the source of energy in the conformational changes that produce muscle contraction but, in general, it is not ATP hydrolysis but the transfer of a phosphoryl, pyrophosphoryl, or adenylyl group from ATP to a substrate or enzyme molecule that couples the energy of ATP breakdown to endergonic transformations of substrates. ■ Through these group transfer reactions, ATP provides the energy for anabolic reactions, including the synthesis of informational molecules, and for the transport of molecules and ions across membranes against concentration gradients and electrical potential gradients. ■ Cells contain other metabolites with large, negative, free energies of hydrolysis, including phosphoenolpyruvate, 1,3-bisphosphoglycerate, and phosphocreatine. These high-energy compounds, like ATP, have a high phosphoryl group transfer potential; they are good donors of the phosphoryl group. Thioesters also have high free energies of hydrolysis. ■ Inorganic polyphosphate, present in all cells, may serve as a reservoir of phosphoryl groups with high group transfer potential. 13.3 Biological Oxidation-Reduction Reactions The transfer of phosphoryl groups is a central feature of metabolism. Equally important is another kind of transfer, electron transfer in oxidation-reduction reac- tions. These reactions involve the loss of electrons by one chemical species, which is thereby oxidized, and the gain of electrons by another, which is reduced. The flow of electrons in oxidation-reduction reactions is respon- sible, directly or indirectly, for all work done by living organisms. In nonphotosynthetic organisms, the sources of electrons are reduced compounds (foods); in photo- synthetic organisms, the initial electron donor is a chem- ical species excited by the absorption of light. The path of electron flow in metabolism is complex. Electrons move from various metabolic intermediates to special- ized electron carriers in enzyme-catalyzed reactions. The carriers in turn donate electrons to acceptors with higher electron affinities, with the release of energy. Cells contain a variety of molecular energy transducers, which convert the energy of electron flow into useful work. We begin our discussion with a description of the general types of metabolic reactions in which electrons are transferred. After considering the theoretical and experimental basis for measuring the energy changes in oxidation reactions in terms of electromotive force, we discuss the relationship between this force, expressed in volts, and the free-energy change, expressed in joules. We conclude by describing the structures and oxidation- reduction chemistry of the most common of the spe- cialized electron carriers, which you will encounter repeatedly in later chapters. The Flow of Electrons Can Do Biological Work Every time we use a motor, an electric light or heater, or a spark to ignite gasoline in a car engine, we use the flow of electrons to accomplish work. In the circuit that powers a motor, the source of electrons can be a bat- tery containing two chemical species that differ in affin- ity for electrons. Electrical wires provide a pathway for electron flow from the chemical species at one pole of the battery, through the motor, to the chemical species at the other pole of the battery. Because the two chem- ical species differ in their affinity for electrons, electrons flow spontaneously through the circuit, driven by a force proportional to the difference in electron affinity, the electromotive force (emf). The electromotive force (typically a few volts) can accomplish work if an ap- propriate energy transducer—in this case a motor—is placed in the circuit. The motor can be coupled to a va- riety of mechanical devices to accomplish useful work. Living cells have an analogous biological “circuit,” with a relatively reduced compound such as glucose as the source of electrons. As glucose is enzymatically ox- idized, the released electrons flow spontaneously through a series of electron-carrier intermediates to an- other chemical species, such as O 2 . This electron flow is exergonic, because O 2 has a higher affinity for elec- trons than do the electron-carrier intermediates. The resulting electromotive force provides energy to a vari- ety of molecular energy transducers (enzymes and other proteins) that do biological work. In the mitochondrion, for example, membrane-bound enzymes couple electron flow to the production of a transmembrane pH differ- ence, accomplishing osmotic and electrical work. The proton gradient thus formed has potential energy, some- times called the proton-motive force by analogy with electromotive force. Another enzyme, ATP synthase in the inner mitochondrial membrane, uses the proton- motive force to do chemical work: synthesis of ATP from ADP and P i as protons flow spontaneously across the membrane. Similarly, membrane-localized enzymes in 13.3 Biological Oxidation-Reduction Reactions 507 E. coli convert electromotive force to proton-motive force, which is then used to power flagellar motion. The principles of electrochemistry that govern en- ergy changes in the macroscopic circuit with a motor and battery apply with equal validity to the molecular processes accompanying electron flow in living cells. We turn now to a discussion of those principles. Oxidation-Reductions Can Be Described as Half-Reactions Although oxidation and reduction must occur together, it is convenient when describing electron transfers to consider the two halves of an oxidation-reduction reac- tion separately. For example, the oxidation of ferrous ion by cupric ion, Fe 2H11001 H11001 Cu 2H11001 34 Fe 3H11001 H11001 Cu H11001 can be described in terms of two half-reactions: (1) Fe 2H11001 34 Fe 3H11001 H11001 e H11002 (2) Cu 2H11001 H11001 e H11002 34 Cu H11001 The electron-donating molecule in an oxidation- reduction reaction is called the reducing agent or reduc- tant; the electron-accepting molecule is the oxidizing agent or oxidant. A given agent, such as an iron cation existing in the ferrous (Fe 2H11001 ) or ferric (Fe 3H11001 ) state, func- tions as a conjugate reductant-oxidant pair (redox pair), just as an acid and corresponding base function as a con- jugate acid-base pair. Recall from Chapter 2 that in acid- base reactions we can write a general equation: proton donor 34 H H11001 H11001 proton acceptor. In redox reactions we can write a similar general equation: electron donor 34 e H11002 H11001 electron acceptor. In the reversible half-reaction (1) above, Fe 2H11001 is the electron donor and Fe 3H11001 is the elec- tron acceptor; together, Fe 2H11001 and Fe 3H11001 constitute a con- jugate redox pair. The electron transfers in the oxidation-reduction reactions of organic compounds are not fundamentally different from those of inorganic species. In Chapter 7 we considered the oxidation of a reducing sugar (an aldehyde or ketone) by cupric ion (see Fig. 7–10a): This overall reaction can be expressed as two half- reactions: (1) (2) 2Cu 2H11001 H11001 2e H11002 H11001 2OH H11002 34 Cu 2 O H11001 H 2 O Because two electrons are removed from the aldehyde carbon, the second half-reaction (the one-electron re- duction of cupric to cuprous ion) must be doubled to balance the overall equation. R C H O H11001 2OH H11002 H11001H110012e H11002 H 2 OR C OH O R C H O H11001H110014OH H11002 2Cu 2H11001 H11001H11001Cu 2 O2H 2 OR C OH O Biological Oxidations Often Involve Dehydrogenation The carbon in living cells exists in a range of oxidation states (Fig. 13–13). When a carbon atom shares an elec- tron pair with another atom (typically H, C, S, N, or O), the sharing is unequal in favor of the more electroneg- ative atom. The order of increasing electronegativity is H H11021 C H11021 S H11021 N H11021 O. In oversimplified but useful terms, the more electronegative atom “owns” the bonding elec- trons it shares with another atom. For example, in methane (CH 4 ), carbon is more electronegative than the four hydrogens bonded to it, and the C atom therefore “owns” all eight bonding electrons (Fig. 13–13). In ethane, the electrons in the COC bond are shared equally, so each C atom owns only seven of its eight bonding electrons. In ethanol, C-1 is less electronega- tive than the oxygen to which it is bonded, and the O atom therefore “owns” both electrons of the COO bond, leaving C-1 with only five bonding electrons. With each formal loss of electrons, the carbon atom has undergone oxidation—even when no oxygen is involved, as in the conversion of an alkane (OCH 2 OCH 2 O) to an alkene (OCHUCHO). In this case, oxidation (loss of elec- trons) is coincident with the loss of hydrogen. In bio- logical systems, oxidation is often synonymous with de- hydrogenation, and many enzymes that catalyze oxidation reactions are dehydrogenases. Notice that the more reduced compounds in Figure 13–13 (top) are richer in hydrogen than in oxygen, whereas the more oxidized compounds (bottom) have more oxygen and less hydrogen. Not all biological oxidation-reduction reactions in- volve carbon. For example, in the conversion of molec- ular nitrogen to ammonia, 6H H11001 H11001 6e H11002 H11001 N 2 n 2NH 3 , the nitrogen atoms are reduced. Electrons are transferred from one molecule (elec- tron donor) to another (electron acceptor) in one of four different ways: 1. Directly as electrons. For example, the Fe 2H11001 /Fe 3H11001 redox pair can transfer an electron to the Cu H11001 /Cu 2H11001 redox pair: Fe 2H11001 H11001 Cu 2H11001 34 Fe 3H11001 H11001 Cu H11001 2. As hydrogen atoms. Recall that a hydrogen atom consists of a proton (H H11001 ) and a single electron (e H11002 ). In this case we can write the general equation AH 2 34 A H11001 2e H11002 H11001 2H H11001 where AH 2 is the hydrogen/electron donor. (Do not mistake the above reaction for an acid dissociation; the H H11001 arises from the removal of a hydrogen atom, H H11001 H11001 e H11002 .) AH 2 and A together constitute a conjugate redox pair (A/AH 2 ), which can reduce another compound B (or redox pair, B/BH 2 ) by transfer of hydrogen atoms: AH 2 H11001 B 34 A H11001 BH 2 Chapter 13 Principles of Bioenergetics508 potential of 0.00 V. When this hydrogen electrode is con- nected through an external circuit to another half-cell in which an oxidized species and its corresponding re- duced species are present at standard concentrations (each solute at 1 M, each gas at 101.3 kPa), electrons tend to flow through the external circuit from the half-cell of 13.3 Biological Oxidation-Reduction Reactions 509 Methane 8 H H HHC Ethane (alkane) 7 H H HC H H HC Ethanol (alcohol) 5 H H HC H H C HO Acetylene (alkyne) 5HHC C Ethene (alkene) 6C C HH HH Acetaldehyde (aldehyde) 3 H H H C O C H Formaldehyde 4 H H C O Carbon monoxide 2C O Carbon dioxide 0O C O Formic acid (carboxylic acid) 2 H H C O O Acetic acid (carboxylic acid) 1 H H H CC H O O Acetone (ketone) 2 H H HC H H C O C H FIGURE 13–13 Oxidation states of carbon in the biosphere. The oxidation states are illustrated with some representative compounds. Focus on the red carbon atom and its bonding electrons. When this carbon is bonded to the less electronegative H atom, both bonding electrons (red) are assigned to the carbon. When carbon is bonded to another carbon, bonding electrons are shared equally, so one of the two electrons is assigned to the red carbon. When the red carbon is bonded to the more electronegative O atom, the bonding electrons are assigned to the oxygen. The number to the right of each compound is the number of electrons “owned” by the red carbon, a rough ex- pression of the oxidation state of that carbon. When the red carbon undergoes oxidation (loses electrons), the number gets smaller. Thus the oxidation state increases from top to bottom of the list. 3. As a hydride ion (:H H11002 ), which has two electrons. This occurs in the case of NAD-linked dehydroge- nases, described below. 4. Through direct combination with oxygen. In this case, oxygen combines with an organic reductant and is covalently incorporated in the product, as in the oxidation of a hydrocarbon to an alcohol: RXCH 3 H11001 H5007 1 2 H5007 O 2 88n RXCH 2 XOH The hydrocarbon is the electron donor and the oxygen atom is the electron acceptor. All four types of electron transfer occur in cells. The neutral term reducing equivalent is commonly used to designate a single electron equivalent participating in an oxidation-reduction reaction, no matter whether this equivalent is an electron per se, a hydrogen atom, or a hy- dride ion, or whether the electron transfer takes place in a reaction with oxygen to yield an oxygenated product. Because biological fuel molecules are usually enzymati- cally dehydrogenated to lose two reducing equivalents at a time, and because each oxygen atom can accept two re- ducing equivalents, biochemists by convention regard the unit of biological oxidations as two reducing equivalents passing from substrate to oxygen. Reduction Potentials Measure Affinity for Electrons When two conjugate redox pairs are together in solu- tion, electron transfer from the electron donor of one pair to the electron acceptor of the other may proceed spontaneously. The tendency for such a reaction de- pends on the relative affinity of the electron acceptor of each redox pair for electrons. The standard reduc- tion potential, EH11543, a measure (in volts) of this affin- ity, can be determined in an experiment such as that described in Figure 13–14. Electrochemists have cho- sen as a standard of reference the half-reaction H H11001 H11001 e H11002 88n H5007 1 2 H5007 H 2 The electrode at which this half-reaction occurs (called a half-cell) is arbitrarily assigned a standard reduction lower standard reduction potential to the half-cell of higher standard reduction potential. By convention, the half-cell with the stronger tendency to acquire electrons is assigned a positive value of EH11034. The reduction potential of a half-cell depends not only on the chemical species present but also on their activities, approximated by their concentrations. About a century ago, Walther Nernst derived an equation that relates standard reduction potential (EH11034) to the reduc- tion potential (E) at any concentration of oxidized and reduced species in the cell: E H11005 EH11034H11001H5007 R n? T H5007 ln (13–4) [electron acceptor] H5007H5007H5007 [electron donor] where R and T have their usual meanings, n is the num- ber of electrons transferred per molecule, and is the Faraday constant (Table 13–1). At 298 K (25 H11034C), this expression reduces to E H11005 EH11034H11001H5007 0.02 n 6V H5007 ln (13–5) Many half-reactions of interest to biochemists in- volve protons. As in the definition of H9004GH11032H11034, biochemists define the standard state for oxidation-reduction reac- tions as pH 7 and express reduction potential as EH11032H11034, the standard reduction potential at pH 7. The standard re- duction potentials given in Table 13–7 and used through- out this book are values for EH11032H11034 and are therefore valid only for systems at neutral pH. Each value represents the potential difference when the conjugate redox pair, at 1 M concentrations and pH 7, is connected with the standard (pH 0) hydrogen electrode. Notice in Table 13–7 that when the conjugate pair 2H H11001 /H 2 at pH 7 is connected with the standard hydrogen electrode (pH 0), electrons tend to flow from the pH 7 cell to the stan- dard (pH 0) cell; the measured EH11032H11034 for the 2H H11001 /H 2 pair is H110020.414 V. Standard Reduction Potentials Can Be Used to Calculate the Free-Energy Change The usefulness of reduction potentials stems from the fact that when E values have been determined for any two half-cells, relative to the standard hydrogen elec- trode, their reduction potentials relative to each other are also known. We can then predict the direction in which electrons will tend to flow when the two half-cells are connected through an external circuit or when com- ponents of both half-cells are present in the same solu- tion. Electrons tend to flow to the half-cell with the more positive E, and the strength of that tendency is pro- portional to the difference in reduction potentials, H9004E. The energy made available by this spontaneous electron flow (the free-energy change for the oxidation- reduction reaction) is proportional to H9004E: H9004G H11005H11002n H9004E or H9004GH11032H11034 H11005 H11002n H9004EH11032H11034 (13–6) Here n represents the number of electrons transferred in the reaction. With this equation we can calculate the free-energy change for any oxidation-reduction reaction from the values of EH11032H11034 in a table of reduction potentials (Table 13–7) and the concentrations of the species par- ticipating in the reaction. Consider the reaction in which acetaldehyde is reduced by the biological electron carrier NADH: Acetaldehyde H11001 NADH H11001 H H11001 88n ethanol H11001 NAD H11001 The relevant half-reactions and their EH11032H11034 values are: (1) Acetaldehyde H11001 2H H11001 H11001 2e H11002 88n ethanol EH11032H11034 H11005 H110020.197 V [electron acceptor] H5007H5007H5007 [electron donor] Chapter 13 Principles of Bioenergetics510 Salt bridge (KCl solution) Reference cell of known emf: the hydrogen electrode in which H 2 gas at 101.3 kPa is equilibrated at the electrode with 1 M H H11001 Test cell containing 1 M concentrations of the oxidized and reduced species of the redox pair to be examined H 2 gas (standard pressure) Device for measuring emf FIGURE 13–14 Measurement of the standard reduction potential (EH11032H11034) of a redox pair. Electrons flow from the test electrode to the ref- erence electrode, or vice versa. The ultimate reference half-cell is the hydrogen electrode, as shown here, at pH 0. The electromotive force (emf) of this electrode is designated 0.00 V. At pH 7 in the test cell, EH11032H11034 for the hydrogen electrode is H110020.414 V. The direction of electron flow depends on the relative electron “pressure” or potential of the two cells. A salt bridge containing a saturated KCl solution provides a path for counter-ion movement between the test cell and the refer- ence cell. From the observed emf and the known emf of the reference cell, the experimenter can find the emf of the test cell containing the redox pair. The cell that gains electrons has, by convention, the more positive reduction potential. (2) NAD H11001 H11001 2H H11001 H11001 2e H11002 88n NADH H11001 H H11001 EH11032H11034 H11005 H110020.320 V By convention, H9004EH11032H11034 is expressed as EH11032H11034 of the electron acceptor minus EH11032H11034 of the electron donor. Because ac- etaldehyde is accepting electrons from NADH in our example, H9004EH11032H11034 H11005 H110020.197 V H11002 (H110020.320 V) H11005 0.123 V, and n is 2. Therefore, H9004GH11032H11034 H11005 H11002n H9004EH11032H11034 H11005 H110022(96.5 kJ/V H11080 mol)(0.123 V) H11005H1100223.7 kJ/mol This is the free-energy change for the oxidation- reduction reaction at pH 7, when acetaldehyde, ethanol, NAD H11001 , and NADH are all present at 1.00 M concentra- tions. If, instead, acetaldehyde and NADH were present at 1.00 M but ethanol and NAD H11001 were present at 0.100 M, the value for H9004G would be calculated as follows. First, the values of E for both reductants are determined (Eqn 13–4): E acetaldehyde H11005 EH11034H11001H5007 R n? T H5007 ln H5007 [ac [ e e t t a h ld a e n h o y l] de] H5007 H11005H110020.197 V H11001 H5007 0.02 2 6V H5007 ln H5007 0 1 .1 .0 0 0 0 H5007 H11005H110020.167 V E NADH H11005 EH11034H11001H5007 R n? T H5007 ln H5007 [ [ N N A A D D H H11001 ] ] H5007 H11005H110020.320 V H11001 H5007 0.02 2 6V H5007 ln H5007 0 1 .1 .0 0 0 0 H5007 H11005H110020.350 V Then H9004E is used to calculate H9004G (Eqn 13–5): H9004E H11005H110020.167 V H11002 (H110020.350) V H11005 0.183 V H9004G H11005H11002n H9004E H11005H110022(96.5 kJ/V H11554 mol)(0.183 V) H11005H1100235.3 kJ/mol 13.3 Biological Oxidation-Reduction Reactions 511 Half-reaction EH11032H11034 (V) H5007 1 2 H5007 O 2 H11001 2H H11001 H11001 2e H11002 88n H 2 O 0.816 Fe 3H11001 H11001 e H11002 88n Fe 2H11001 0.771 NO 3 H11002 H11001 2H H11001 H11001 2e H11002 88n NO 2 H11002 H11001 H 2 O 0.421 Cytochrome f (Fe 3H11001 ) H11001 e H11002 88n cytochrome f (Fe 2H11001 ) 0.365 Fe(CN) 6 3H11002 (ferricyanide) H11001 e H11002 88n Fe(CN) 6 4H11002 0.36 Cytochrome a 3 (Fe 3H11001 ) H11001 e H11002 88n cytochrome a 3 (Fe 2H11001 ) 0.35 O 2 H11001 2H H11001 H11001 2e H11002 88n H 2 O 2 0.295 Cytochrome a (Fe 3H11001 ) H11001 e H11002 88n cytochrome a (Fe 2H11001 ) 0.29 Cytochrome c (Fe 3H11001 ) H11001 e H11002 88n cytochrome c (Fe 2H11001 ) 0.254 Cytochrome c 1 (Fe 3H11001 ) H11001 e H11002 88n cytochrome c 1 (Fe 2H11001 ) 0.22 Cytochrome b (Fe 3H11001 ) H11001 e H11002 88n cytochrome b (Fe 2H11001 ) 0.077 Ubiquinone H11001 2H H11001 H11001 2e H11002 88n ubiquinol H11001 H 2 0.045 Fumarate 2H11002 H11001 2H H11001 H11001 2e H11002 88n succinate 2H11002 0.031 2H H11001 H11001 2e H11002 88n H 2 (at standard conditions, pH 0) 0.000 Crotonyl-CoA H11001 2H H11001 H11001 2e H11002 88n butyryl-CoA H110020.015 Oxaloacetate 2H11002 H11001 2H H11001 H11001 2e H11002 88n malate 2H11002 H110020.166 Pyruvate H11002 H11001 2H H11001 H11001 2e H11002 88n lactate H11002 H110020.185 Acetaldehyde H11001 2H H11001 H11001 2e H11002 88n ethanol H110020.197 FAD H11001 2H H11001 H11001 2e H11002 88n FADH 2 H110020.219* Glutathione H11001 2H H11001 H11001 2e H11002 88n 2 reduced glutathione H110020.23 S H11001 2H H11001 H11001 2e H11002 88n H 2 S H110020.243 Lipoic acid H11001 2H H11001 H11001 2e H11002 88n dihydrolipoic acid H110020.29 NAD H11001 H11001 H H11001 H11001 2e H11002 88n NADH H110020.320 NADP H11001 H11001 H H11001 H11001 2e H11002 88n NADPH H110020.324 Acetoacetate H11001 2H H11001 H11001 2e H11002 88n H9252-hydroxybutyrate H110020.346 H9251-Ketoglutarate H11001 CO 2 H11001 2H H11001 H11001 2e H11002 88n isocitrate H110020.38 2H H11001 H11001 2e H11002 88n H 2 (at pH 7) H110020.414 Ferredoxin (Fe 3H11001 ) H11001 e H11002 88n ferredoxin (Fe 2H11001 ) H110020.432 Standard Reduction Potentials of Some Biologically Important Half-Reactions, at pH 7.0 and 25 H11034C (298 K) TABLE 13–7 Source: Data mostly from Loach, P.A. (1976) In Handbook of Biochemistry and Molecular Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. I, pp. 122–130, CRC Press, Boca Raton, FL. * This is the value for free FAD; FAD bound to a specific flavoprotein (for example succinate dehydrogenase) has a different EH11032H11034 that depends on its protein environments. It is thus possible to calculate the free-energy change for any biological redox reaction at any concentrations of the redox pairs. Cellular Oxidation of Glucose to Carbon Dioxide Requires Specialized Electron Carriers The principles of oxidation-reduction energetics de- scribed above apply to the many metabolic reactions that involve electron transfers. For example, in many organ- isms, the oxidation of glucose supplies energy for the production of ATP. The complete oxidation of glucose: C 6 H 12 O 6 H11001 6O 2 8n 6CO 2 H11001 6H 2 O has a H9004GH11032H11034 of H110022,840 kJ/mol. This is a much larger re- lease of free energy than is required for ATP synthesis (50 to 60 kJ/mol; see Box 13–1). Cells convert glucose to CO 2 not in a single, high-energy-releasing reaction, but rather in a series of controlled reactions, some of which are oxidations. The free energy released in these oxidation steps is of the same order of magnitude as that required for ATP synthesis from ADP, with some energy to spare. Electrons removed in these oxidation steps are transferred to coenzymes specialized for carrying elec- trons, such as NAD H11001 and FAD (described below). A Few Types of Coenzymes and Proteins Serve as Universal Electron Carriers The multitude of enzymes that catalyze cellular oxida- tions channel electrons from their hundreds of different substrates into just a few types of universal electron car- riers. The reduction of these carriers in catabolic processes results in the conservation of free energy re- leased by substrate oxidation. NAD H11001 , NADP H11001 , FMN, and FAD are water-soluble coenzymes that undergo re- versible oxidation and reduction in many of the electron- transfer reactions of metabolism. The nucleotides NAD H11001 and NADP H11001 move readily from one enzyme to another; the flavin nucleotides FMN and FAD are usually very tightly bound to the enzymes, called flavoproteins, for which they serve as prosthetic groups. Lipid-soluble quinones such as ubiquinone and plastoquinone act as electron carriers and proton donors in the nonaqueous environment of membranes. Iron-sulfur proteins and cy- tochromes, which have tightly bound prosthetic groups that undergo reversible oxidation and reduction, also serve as electron carriers in many oxidation-reduction reactions. Some of these proteins are water-soluble, but others are peripheral or integral membrane proteins (see Fig. 11–6). We conclude this chapter by describing some chem- ical features of nucleotide coenzymes and some of the enzymes (dehydrogenases and flavoproteins) that use them. The oxidation-reduction chemistry of quinones, iron-sulfur proteins, and cytochromes is discussed in Chapter 19. NADH and NADPH Act with Dehydrogenases as Soluble Electron Carriers Nicotinamide adenine dinucleotide (NAD H11001 in its oxi- dized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP H11001 ) are composed of two nucleotides joined through their phosphate groups by a phosphoanhydride bond (Fig. 13–15a). Because the nicotinamide ring resembles pyridine, these compounds are sometimes called pyridine nucleotides. The vita- min niacin is the source of the nicotinamide moiety in nicotinamide nucleotides. Both coenzymes undergo reversible reduction of the nicotinamide ring (Fig. 13–15). As a substrate mol- ecule undergoes oxidation (dehydrogenation), giving up two hydrogen atoms, the oxidized form of the nucleotide (NAD H11001 or NADP H11001 ) accepts a hydride ion (:H H11002 , the equivalent of a proton and two electrons) and is trans- formed into the reduced form (NADH or NADPH). The second proton removed from the substrate is released to the aqueous solvent. The half-reaction for each type of nucleotide is therefore NAD H11001 H11001 2e H11002 H11001 2H H11001 8n NADH H11001 H H11001 NADP H11001 H11001 2e H11002 H11001 2H H11001 8n NADPH H11001 H H11001 Reduction of NAD H11001 or NADP H11001 converts the benzenoid ring of the nicotinamide moiety (with a fixed positive charge on the ring nitrogen) to the quinonoid form (with no charge on the nitrogen). Note that the reduced nu- cleotides absorb light at 340 nm; the oxidized forms do not (Fig. 13–15b). The plus sign in the abbreviations NAD H11001 and NADP H11001 does not indicate the net charge on these molecules (they are both negatively charged); rather, it indicates that the nicotinamide ring is in its oxidized form, with a positive charge on the nitrogen atom. In the abbreviations NADH and NADPH, the “H” denotes the added hydride ion. To refer to these nu- cleotides without specifying their oxidation state, we use NAD and NADP. The total concentration of NAD H11001 H11001 NADH in most tissues is about 10 H110025 M; that of NADP H11001 H11001 NADPH is about 10 H110026 M. In many cells and tissues, the ratio of NAD H11001 (oxidized) to NADH (reduced) is high, favoring hydride transfer from a substrate to NAD H11001 to form NADH. By contrast, NADPH (reduced) is generally pres- ent in greater amounts than its oxidized form, NADP H11001 , favoring hydride transfer from NADPH to a substrate. This reflects the specialized metabolic roles of the two coenzymes: NAD H11001 generally functions in oxidations— usually as part of a catabolic reaction; and NADPH is the usual coenzyme in reductions—nearly always as part of an anabolic reaction. A few enzymes can use ei- ther coenzyme, but most show a strong preference for one over the other. The processes in which these two cofactors function are also segregated in specific or- ganelles of eukaryotic cells: oxidations of fuels such as pyruvate, fatty acids, and H9251-keto acids derived from Chapter 13 Principles of Bioenergetics512 amino acids occur in the mitochondrial matrix, whereas reductive biosynthesis processes such as fatty acid syn- thesis take place in the cytosol. This functional and spa- tial specialization allows a cell to maintain two distinct pools of electron carriers, with two distinct functions. More than 200 enzymes are known to catalyze re- actions in which NAD H11001 (or NADP H11001 ) accepts a hydride ion from a reduced substrate, or NADPH (or NADH) do- nates a hydride ion to an oxidized substrate. The gen- eral reactions are AH 2 H11001 NAD H11001 8n A H11001 NADH H11001 H H11001 A H11001 NADPH H11001 H H11001 8n AH 2 H11001 NADP H11001 where AH 2 is the reduced substrate and A the oxidized substrate. The general name for an enzyme of this type is oxidoreductase; they are also commonly called de- hydrogenases. For example, alcohol dehydrogenase catalyzes the first step in the catabolism of ethanol, in which ethanol is oxidized to acetaldehyde: CH 3 CH 2 OH H11001 NAD H11001 8n CH 3 CHO H11001 NADH H11001 H H11001 Ethanol Acetaldehyde Notice that one of the carbon atoms in ethanol has lost a hydrogen; the compound has been oxidized from an alcohol to an aldehyde (refer again to Fig. 13–13 for the oxidation states of carbon). When NAD H11001 or NADP H11001 is reduced, the hydride ion could in principle be transferred to either side of the nicotinamide ring: the front (A side) or the back (B side), as represented in Figure 13–15a. Studies with iso- topically labeled substrates have shown that a given en- zyme catalyzes either an A-type or a B-type transfer, but not both. For example, yeast alcohol dehydrogenase and lactate dehydrogenase of vertebrate heart transfer a hy- dride ion to (or remove a hydride ion from) the A side of the nicotinamide ring; they are classed as type A de- hydrogenases to distinguish them from another group of enzymes that transfer a hydride ion to (or remove a hydride ion from) the B side of the nicotinamide ring (Table 13–8). The specificity for one side or another can be very striking; lactate dehydrogenase, for example, prefers the A side over the B side by a factor of 5 H11003 10 7 ! Most dehydrogenases that use NAD or NADP bind the cofactor in a conserved protein domain called the Rossmann fold (named for Michael Rossmann, who de- duced the structure of lactate dehydrogenase and first described this structural motif). The Rossmann fold typ- ically consists of a six-stranded parallel H9252 sheet and four associated H9251 helices (Fig. 13–16). The association between a dehydrogenase and NAD or NADP is relatively loose; the coenzyme readily diffuses from one enzyme to another, acting as a water-soluble 13.3 Biological Oxidation-Reduction Reactions 513 C A CH 2 POP OH H O H H11001 H N OH R NH 2 PO O H11002 O O O P O CH 2 OH H H H H OH O O H11002 O NH 2 B side N (b) N N H H N or NADH ? N H11001 C NH 2 B O HH C A R NH 2 ? N B O H In NADP H11001 this hydroxyl group is esterified with phosphate. 2H H11001 2e H11002 H Absorbance NAD H11545 (reduced) A side H H11001 (oxidized) 1.0 Wavelength (nm) 0.0 220 240 260 280 300 320 340 360 380 Oxidized (NAD H11001 ) Reduced (NADH) (a) 0.6 0.4 0.2 0.8 B H HH Adenine O FIGURE 13–15 NAD and NADP. (a) Nicotinamide adenine dinu- cleotide, NAD H11001 , and its phosphorylated analog NADP H11001 undergo re- duction to NADH and NADPH, accepting a hydride ion (two elec- trons and one proton) from an oxidizable substrate. The hydride ion is added to either the front (the A side) or the back (the B side) of the planar nicotinamide ring (see Table 13–8). (b) The UV absorption spec- tra of NAD H11001 and NADH. Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340 nm. The pro- duction of NADH during an enzyme-catalyzed reaction can be con- veniently followed by observing the appearance of the absorbance at 340 nm (the molar extinction coefficient H9255 340 H11005 6,200 M H110021 cm H110021 ). carrier of electrons from one metabolite to another. For example, in the production of alcohol during fermenta- tion of glucose by yeast cells, a hydride ion is removed from glyceraldehyde 3-phosphate by one enzyme (glyc- eraldehyde 3-phosphate dehydrogenase, a type B en- zyme) and transferred to NAD H11001 . The NADH produced then leaves the enzyme surface and diffuses to another enzyme (alcohol dehydrogenase, a type A enzyme), which transfers a hydride ion to acetaldehyde, produc- ing ethanol: (1) Glyceraldehyde 3-phosphate H11001 NAD H11001 8n 3-phosphoglycerate H11001 NADH H11001 H H11001 (2) Acetaldehyde H11001 NADH H11001 H H11001 8n ethanol H11001 NAD H11001 Sum: Glyceraldehyde 3-phosphate H11001 acetaldehyde 8n 3-phosphoglycerate H11001 ethanol Notice that in the overall reaction there is no net pro- duction or consumption of NAD H11001 or NADH; the coen- zymes function catalytically and are recycled repeatedly without a net change in the concentration of NAD H11001 H11001 NADH. Dietary Deficiency of Niacin, the Vitamin Form of NAD and NADP, Causes Pellagra The pyridine-like rings of NAD and NADP are de- rived from the vitamin niacin (nicotinic acid; Fig. 13–17), which is synthesized from tryptophan. Humans generally cannot synthesize niacin in sufficient quanti- ties, and this is especially so for those with diets low in tryptophan (maize, for example, has a low tryptophan content). Niacin deficiency, which affects all the NAD(P)-dependent dehydrogenases, causes the serious human disease pellagra (Italian for “rough skin”) and a related disease in dogs, blacktongue. These diseases are characterized by the “three Ds”: dermatitis, diarrhea, and dementia, followed in many cases by death. A century ago, pellagra was a common human disease; in the south- ern United States, where maize was a dietary staple, about 100,000 people were afflicted and about 10,000 died between 1912 and 1916. In 1920 Joseph Goldberger showed pellagra to be caused by a dietary insufficiency, and in 1937 Frank Strong, D. Wayne Wolley, and Conrad Elvehjem identified niacin as the curative agent for blacktongue. Supplementation of the human diet with this inexpensive compound led to the eradication of pel- lagra in the populations of the developed world—with one significant exception. Pellagra is still found among alcoholics, whose intestinal absorption of niacin is much Chapter 13 Principles of Bioenergetics514 Stereochemical specificity for nicotinamide Enzyme Coenzyme ring (A or B) Text page(s) Isocitrate dehydrogenase NAD H11001 A XXX–XXX H9251-Ketoglutarate dehydrogenase NAD H11001 B XXX Glucose 6-phosphate dehydrogenase NADP H11001 B XXX Malate dehydrogenase NAD H11001 A XXX Glutamate dehydrogenase NAD H11001 or NADP H11001 B XXX Glyceraldehyde 3-phosphate dehydrogenase NAD H11001 B XXX Lactate dehydrogenase NAD H11001 A XXX Alcohol dehydrogenase NAD H11001 A XXX TABLE 13–8 Stereospecificity of Dehydrogenases That Employ NAD H11545 or NADP H11545 as Coenzymes FIGURE 13–16 The nucleotide binding domain of the enzyme lac- tate dehydrogenase. (a) The Rossmann fold is a structural motif found in the NAD-binding site of many dehydrogenases. It consists of a six-stranded parallel H9252 sheet and four H9251 helices; inspection reveals the arrangement to be a pair of structurally similar H9252-H9251-H9252-H9251-H9252 motifs. (b) The dinucleotide NAD binds in an extended conformation through hydrogen bonds and salt bridges (derived from PDB ID 3LDH). reduced, and whose caloric needs are often met with dis- tilled spirits that are virtually devoid of vitamins, in- cluding niacin. In a few places, including the Deccan Plateau in India, pellagra still occurs, especially among the poor. ■ Flavin Nucleotides Are Tightly Bound in Flavoproteins Flavoproteins (Table 13–9) are enzymes that catalyze oxidation-reduction reactions using either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as coenzyme (Fig. 13–18). These coenzymes, the flavin nucleotides, are derived from the vitamin ri- boflavin. The fused ring structure of flavin nucleotides (the isoalloxazine ring) undergoes reversible reduction, accepting either one or two electrons in the form of one or two hydrogen atoms (each atom an electron plus a proton) from a reduced substrate. The fully reduced forms are abbreviated FADH 2 and FMNH 2 . When a fully oxidized flavin nucleotide accepts only one electron (one hydrogen atom), the semiquinone form of the isoal- loxazine ring is produced, abbreviated FADH ? and FMNH ? . Because flavoproteins can participate in either one- or two-electron transfers, this class of proteins is involved in a greater diversity of reactions than the NAD (P)-linked dehydrogenases. Like the nicotinamide coenzymes (Fig. 13–15), the flavin nucleotides undergo a shift in a major absorption band on reduction. Flavoproteins that are fully reduced (two electrons accepted) generally have an absorption maximum near 360 nm. When partially reduced (one electron), they acquire another absorption maximum at about 450 nm; when fully oxidized, the flavin has max- ima at 370 and 440 nm. The intermediate radical form, reduced by one electron, has absorption maxima at 380, 480, 580, and 625 nm. These changes can be used to as- say reactions involving a flavoprotein. The flavin nucleotide in most flavoproteins is bound rather tightly to the protein, and in some enzymes, such as succinate dehydrogenase, it is bound covalently. Such tightly bound coenzymes are properly called prosthetic groups. They do not transfer electrons by diffusing from one enzyme to another; rather, they provide a means by which the flavoprotein can temporarily hold electrons while it catalyzes electron transfer from a reduced sub- strate to an electron acceptor. One important feature of the flavoproteins is the variability in the standard re- duction potential (EH11032H11034) of the bound flavin nucleotide. Tight as- sociation between the enzyme and prosthetic group confers on the flavin ring a reduction potential typical of that particular flavopro- tein, sometimes quite different from the reduction potential of the free flavin nucleotide. FAD bound to succinate dehydrogenase, for example, has an EH11032H11034 close to 0.0 V, compared with H110020.219 V for free FAD; EH11032H11034 for other flavoproteins ranges from H110020.40 V to H110010.06 V. 13.3 Biological Oxidation-Reduction Reactions 515 O O H11002 H9013 C O H9013 NH 2 H11001 NH 3 CH 3 CH 2 CH COO H11002 C H9013 H9013 H9013 H9007 C Niacin (nicotinic acid) Nicotine Nicotinamide Tryptophan FIGURE 13–17 Structures of niacin (nicotinic acid) and its deriva- tive nicotinamide. The biosynthetic precursor of these compounds is tryptophan. In the laboratory, nicotinic acid was first produced by ox- idation of the natural product nicotine—thus the name. Both nicotinic acid and nicotinamide cure pellagra, but nicotine (from cigarettes or elsewhere) has no curative activity. Frank Strong, 1908–1993 D. Wayne Woolley, 1914–1966 Conrad Elvehjem, 1901–1962 Flavin Text Enzyme nucleotide page(s) Acyl–CoA dehydrogenase FAD XXX Dihydrolipoyl dehydrogenase FAD XXX Succinate dehydrogenase FAD XXX Glycerol 3-phosphate dehydrogenase FAD XXX Thioredoxin reductase FAD XXX–XXX NADH dehydrogenase (Complex I) FMN XXX Glycolate oxidase FMN XXX TABLE 13–9 Some Enzymes (Flavoproteins) That Employ Flavin Nucleotide Coenzymes OH N H H OH R H NH HCOH N O O N HCOH HCOH P O O O P O O H N N N O NH N NN O R NH N NN O H O H H FAD FMN H11001 ? H11002 O H11002 O Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) CH 2 NH 2 CH 2 CH 2 CH 3 CH 3 isoalloxazine ring H H11001 e H11002 H11001H H11001 e H11002 CH 3 CH 3 N H11001 O H11002 FADH ? (FMNH ? ) (semiquinone) CH 3 CH 3 FADH 2 (FMNH 2 ) (fully reduced) Flavoproteins are often very complex; some have, in ad- dition to a flavin nucleotide, tightly bound inorganic ions (iron or molybdenum, for example) capable of partici- pating in electron transfers. Certain flavoproteins act in a quite different role as light receptors. Cryptochromes are a family of flavo- proteins, widely distributed in the eukaryotic phyla, that mediate the effects of blue light on plant development and the effects of light on mammalian circadian rhythms (oscillations in physiology and biochemistry, with a 24-hour period). The cryptochromes are homologs of another family of flavoproteins, the photolyases. Found in both prokaryotes and eukaryotes, photolyases use the energy of absorbed light to repair chemical defects in DNA. We examine the function of flavoproteins as elec- tron carriers in Chapter 19, when we consider their roles in oxidative phosphorylation (in mitochondria) and pho- tophosphorylation (in chloroplasts), and we describe the photolyase reactions in Chapter 25. SUMMARY 13.3 Biological Oxidation-Reduction Reactions ■ In many organisms, a central energy-conserving process is the stepwise oxidation of glucose to CO 2 , in which some of the energy of oxidation is conserved in ATP as electrons are passed to O 2 . ■ Biological oxidation-reduction reactions can be described in terms of two half-reactions, each with a characteristic standard reduction potential, EH11032H11034. ■ When two electrochemical half-cells, each containing the components of a half-reaction, are connected, electrons tend to flow to the half-cell with the higher reduction potential. The strength of this tendency is proportional to the difference between the two reduction potentials (H9004E) and is a function of the concentrations of oxidized and reduced species. ■ The standard free-energy change for an oxidation-reduction reaction is directly proportional to the difference in standard reduction potentials of the two half-cells: H9004GH11032H11034 H11005 H11002n H9004EH11032H11034. ■ Many biological oxidation reactions are dehydrogenations in which one or two hydrogen atoms (H H11001 H11001 e H11002 ) are transferred from a substrate to a hydrogen acceptor. Oxidation-reduction reactions in living cells involve specialized electron carriers. ■ NAD and NADP are the freely diffusible coenzymes of many dehydrogenases. Both NAD H11001 and NADP H11001 accept two electrons and Chapter 13 Principles of Bioenergetics516 FIGURE 13–18 Structures of oxidized and reduced FAD and FMN. FMN consists of the structure above the dashed line on the FAD (ox- idized form). The flavin nucleotides accept two hydrogen atoms (two electrons and two protons), both of which appear in the flavin ring system. When FAD or FMN accepts only one hydrogen atom, the semi- quinone, a stable free radical, forms. one proton. NAD and NADP are bound to dehydrogenases in a widely conserved structural motif called the Rossmann fold. ■ FAD and FMN, the flavin nucleotides, serve as tightly bound prosthetic groups of flavoproteins. They can accept either one or two electrons. Flavoproteins also serve as light receptors in cryptochromes and photolyases. Chapter 13 Further Reading 517 Key Terms autotroph XXX heterotroph XXX metabolism XXX metabolic pathways XXX metabolite XXX intermediary metabolism XXX catabolism XXX anabolism XXX standard transformed constants XXX phosphorylation potential (H9004G p ) XXX thioester XXX adenylylation XXX inorganic pyrophosphatase XXX nucleoside diphosphate kinase XXX adenylate kinase XXX creatine kinase XXX phosphagens XXX polyphosphate kinase-1, -2 XXX electromotive force (emf) XXX conjugate redox pair XXX dehydrogenation XXX dehydrogenases XXX reducing equivalent XXX standard reduction potential (EH11541H11034) XXX pyridine nucleotide XXX oxidoreductase XXX flavoprotein XXX flavin nucleotides XXX cryptochrome XXX photolyase XXX Terms in bold are defined in the glossary. Further Reading Bioenergetics and Thermodynamics Atkins, P.W. (1984) The Second Law, Scientific American Books, Inc., New York. A well-illustrated and elementary discussion of the second law and its implications. Becker, W.M. (1977) Energy and the Living Cell: An Introduc- tion to Bioenergetics, J. B. Lippincott Company, Philadelphia. A clear introductory account of cellular metabolism, in terms of energetics. Bergethon, P.R. (1998) The Physical Basis of Biochemistry, Springer Verlag, New York. Chapters 11 through 13 of this book, and the books by Tinoco et al. and van Holde et al. (below), are excellent general refer- ences for physical biochemistry, with good discussions of the applications of thermodynamics to biochemistry. Edsall, J.T. & Gutfreund, H. (1983) Biothermodynamics: The Study of Biochemical Processes at Equilibrium, John Wiley & Sons, Inc., New York. Harold, F.M. (1986) The Vital Force: A Study of Bioenergetics, W. H. Freeman and Company, New York. A beautifully clear discussion of thermodynamics in biological processes. Harris, D.A. (1995) Bioenergetics at a Glance, Blackwell Science, Oxford. A short, clearly written account of cellular energetics, including introductory chapters on thermodynamics. Loewenstein, W.R. (1999) The Touchstone of Life: Molecular Information, Cell Communication, and the Foundations of Life, Oxford University Press, New York. Beautifully written discussion of the relationship between entropy and information. Morowitz, H.J. (1978) Foundations of Bioenergetics, Academic Press, Inc., New York. [Out of print.] Clear, rigorous description of thermodynamics in biology. Nicholls, D.G. & Ferguson, S.J. (2002) Bioenergetics 3, Academic Press, Inc., New York. Clear, well-illustrated intermediate-level discussion of the theory of bioenergetics and the mechanisms of energy transductions. Tinoco, I., Jr., Sauer, K., & Wang, J.C. (1996) Physical Chem- istry: Principles and Applications in Biological Sciences, 3rd edn, Prentice-Hall, Inc., Upper Saddle River, NJ. Chapters 2 through 5 cover thermodynamics. van Holde, K.E., Johnson, W.C., & Ho, P.S. (1998) Principles of Physical Biochemistry, Prentice-Hall, Inc., Upper Saddle River, NJ. Chapters 2 and 3 are especially relevant. Phosphoryl Group Transfers and ATP Alberty, R.A. (1994) Biochemical thermodynamics. Biochim. Biophys. Acta 1207, 1–11. Explains the distinction between biochemical and chemical equations, and the calculation and meaning of transformed thermodynamic properties for ATP and other phosphorylated compounds. Bridger, W.A. & Henderson, J.F. (1983) Cell ATP, John Wiley & Sons, Inc., New York. The chemistry of ATP, its role in metabolic regulation, and its catabolic and anabolic roles. Frey, P.A. & Arabshahi, A. (1995) Standard free-energy change for the hydrolysis of the H9251–H9252-phosphoanhydride bridge in ATP. Biochemistry 34, 11,307–11,310. Chapter 13 Principles of Bioenergetics518 Hanson, R.W. (1989) The role of ATP in metabolism. Biochem. Educ. 17, 86–92. Excellent summary of the chemistry and biology of ATP. Kornberg, A. (1999) Inorganic polyphosphate: a molecule of many functions. Annu. Rev. Biochem. 68, 89–125. Lipmann, F. (1941) Metabolic generation and utilization of phosphate bond energy. Adv. Enzymol. 11, 99–162. The classic description of the role of high-energy phosphate compounds in biology. Pullman, B. & Pullman, A. (1960) Electronic structure of energy-rich phosphates. Radiat. Res., Suppl. 2, 160–181. An advanced discussion of the chemistry of ATP and other “energy-rich” compounds. Veech, R.L., Lawson, J.W.R., Cornell, N.W., & Krebs, H.A. (1979) Cytosolic phosphorylation potential. J. Biol. Chem. 254, 6538–6547. Experimental determination of ATP, ADP, and P i concentrations in brain, muscle, and liver, and a discussion of the problems in determining the real free-energy change for ATP synthesis in cells. Westheimer, F.H. (1987) Why nature chose phosphates. Science 235, 1173–1178. A chemist’s description of the unique suitability of phosphate esters and anhydrides for metabolic transformations. Biological Oxidation-Reduction Reactions Cashmore, A.R., Jarillo, J.A., Wu, Y.J., & Liu D. (1999) Cryptochromes: blue light receptors for plants and animals. Science 284, 760–765. Dolphin, D., Avramovic, O., & Poulson, R. (eds) (1987) Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and Medical Aspects, John Wiley & Sons, Inc., New York. An excellent two-volume collection of authoritative reviews. Among the most useful are the chapters by Kaplan, Westheimer, Veech, and Ohno and Ushio. Fraaije, M.W. & Mattevi, A. (2000) Flavoenzymes: diverse cata- lysts with recurrent features. Trends Biochem. Sci. 25, 126–132. Massey, V. (1994) Activation of molecular oxygen by flavins and flavoproteins. J. Biol. Chem. 269, 22,459–22,462. A short review of the chemistry of flavin–oxygen interactions in flavoproteins. Rees, D.C. (2002) Great metalloclusters in enzymology. Annu. Rev. Biochem. 71, 221–246. Advanced review of the types of metal ion clusters found in enzymes and their modes of action. Williams, R.E. & Bruce, N.C. (2002) New uses for an old enzyme—the old yellow enzyme family of flavoenzymes. Microbiology 148, 1607–1614. 1. Entropy Changes during Egg Development Con- sider a system consisting of an egg in an incubator. The white and yolk of the egg contain proteins, carbohydrates, and lipids. If fertilized, the egg is transformed from a single cell to a complex organism. Discuss this irreversible process in terms of the entropy changes in the system, surroundings, and universe. Be sure that you first clearly define the system and surroundings. 2. Calculation of H9004GH11541H11543 from an Equilibrium Constant Calculate the standard free-energy changes of the following metabolically important enzyme-catalyzed reactions at 25 H11034C and pH 7.0, using the equilibrium constants given. aspartate aminotransferase (a) Glutamate H11001 oxaloacetate 3::::::::::::4 aspartate H11001H9251-ketoglutarate KH11032 eq H11005 6.8 triose phosphate isomerase (b) Dihydroxyacetone phosphate 3:::::::::::4 glyceraldehyde 3-phosphate KH11032 eq H11005 0.0475 phosphofructokinase (c) Fructose 6-phosphate H11001 ATP 3:::::::::::::::4 fructose 1,6-bisphosphate H11001 ADP KH11032 eq H11005 254 3. Calculation of the Equilibrium Constant from H9004GH11032H11034 Calculate the equilibrium constants KH11032 eq for each of the fol- lowing reactions at pH 7.0 and 25 H11034C, using the H9004GH11032H11034 values in Table 13–4. (a) Glucose 6-phosphate H11001 H 2 O glucose H11001 P i (b) Lactose H11001 H 2 O glucose H11001 galactose (c) Malate fumarate H11001 H 2 O 4. Experimental Determination of KH11541 eq and H9004GH11541H11543 If a 0.1 M solution of glucose 1-phosphate is incubated with a catalytic amount of phosphoglucomutase, the glucose 1-phosphate is transformed to glucose 6-phosphate. At equilibrium, the con- centrations of the reaction components are Glucose 1-phosphate 34 glucose 6-phosphate 4.5 H11003 10 H110023 M 9.6 H11003 10 H110022 M Calculate KH11032 eq and H9004GH11032H11034 for this reaction at 25 H11034C. 5. Experimental Determination of H9004GH11541H11543 for ATP Hy- drolysis A direct measurement of the standard free-energy change associated with the hydrolysis of ATP is technically demanding because the minute amount of ATP remaining at equilibrium is difficult to measure accurately. The value of H9004GH11032H11034 can be calculated indirectly, however, from the equilib- fumarase 3::::::4 b-galactosidase 3::::::::::4 glucose 6-phosphatase 3::::::::::4 Problems Chapter 13 Problems 519 rium constants of two other enzymatic reactions having less favorable equilibrium constants: Glucose 6-phosphate H11001 H 2 O 8n glucose H11001 P i KH11032 eq H11005 270 ATP H11001 glucose 8n ADP H11001 glucose 6-phosphate KH11032 eq H11005 890 Using this information, calculate the standard free energy of hydrolysis of ATP at 25 H11034C. 6. Difference between H9004GH11541H11543 and H9004G Consider the fol- lowing interconversion, which occurs in glycolysis (Chapter 14): Fructose 6-phosphate 34 glucose 6-phosphate KH11032 eq H11005 1.97 (a) What is H9004GH11032H11034 for the reaction (at 25 H11034C)? (b) If the concentration of fructose 6-phosphate is ad- justed to 1.5 M and that of glucose 6-phosphate is adjusted to 0.50 M, what is H9004G? (c) Why are H9004GH11032H11034 and H9004G different? 7. Dependence of H9004G on pH The free energy released by the hydrolysis of ATP under standard conditions at pH 7.0 is H1100230.5 kJ/mol. If ATP is hydrolyzed under standard con- ditions but at pH 5.0, is more or less free energy released? Explain. 8. The H9004GH11541H11543 for Coupled Reactions Glucose 1-phos- phate is converted into fructose 6-phosphate in two succes- sive reactions: Glucose 1-phosphate 88n glucose 6-phosphate Glucose 6-phosphate 88n fructose 6-phosphate Using the H9004GH11032H11034 values in Table 13–4, calculate the equilibrium constant, KH11032 eq , for the sum of the two reactions at 25 H11034C: Glucose 1-phosphate 88n fructose 6-phosphate 9. Strategy for Overcoming an Unfavorable Reaction: ATP-Dependent Chemical Coupling The phosphoryla- tion of glucose to glucose 6-phosphate is the initial step in the catabolism of glucose. The direct phosphorylation of glu- cose by P i is described by the equation Glucose H11001 P i 88n glucose 6-phosphate H11001 H 2 O H9004GH11032H11034 H11005 13.8 kJ/mol (a) Calculate the equilibrium constant for the above re- action. In the rat hepatocyte the physiological concentrations of glucose and P i are maintained at approximately 4.8 mM. What is the equilibrium concentration of glucose 6-phosphate obtained by the direct phosphorylation of glucose by P i ? Does this reaction represent a reasonable metabolic step for the catabolism of glucose? Explain. (b) In principle, at least, one way to increase the con- centration of glucose 6-phosphate is to drive the equilibrium reaction to the right by increasing the intracellular concen- trations of glucose and P i . Assuming a fixed concentration of P i at 4.8 mM, how high would the intracellular concentration of glucose have to be to give an equilibrium concentration of glucose 6-phosphate of 250 H9262M (the normal physiological con- centration)? Would this route be physiologically reasonable, given that the maximum solubility of glucose is less than 1 M? (c) The phosphorylation of glucose in the cell is coupled to the hydrolysis of ATP; that is, part of the free energy of ATP hydrolysis is used to phosphorylate glucose: (1) Glucose H11001 P i 8n glucose 6-phosphate H11001 H 2 O H9004GH11032H11034 H11005 13.8 kJ/mol (2) ATP H11001 H 2 O 8n ADP H11001 P i H9004GH11032H11034 H11005 H1100230.5 kJ/mol Sum: Glucose H11001 ATP 8n glucose 6-phosphate H11001 ADP Calculate KH11032 eq for the overall reaction. For the ATP-dependent phosphorylation of glucose, what concentration of glucose is needed to achieve a 250 H9262M intracellular concentration of glu- cose 6-phosphate when the concentrations of ATP and ADP are 3.38 mM and 1.32 mM, respectively? Does this coupling process provide a feasible route, at least in principle, for the phosphorylation of glucose in the cell? Explain. (d) Although coupling ATP hydrolysis to glucose phos- phorylation makes thermodynamic sense, we have not yet specified how this coupling is to take place. Given that cou- pling requires a common intermediate, one conceivable route is to use ATP hydrolysis to raise the intracellular concentra- tion of P i and thus drive the unfavorable phosphorylation of glucose by P i . Is this a reasonable route? (Think about the solubility products of metabolic intermediates.) (e) The ATP-coupled phosphorylation of glucose is cat- alyzed in hepatocytes by the enzyme glucokinase. This en- zyme binds ATP and glucose to form a glucose-ATP-enzyme complex, and the phosphoryl group is transferred directly from ATP to glucose. Explain the advantages of this route. 10. Calculations of H9004GH11541H11543 for ATP-Coupled Reactions From data in Table 13–6 calculate the H9004GH11032H11034 value for the reactions (a) Phosphocreatine H11001 ADP 8n creatine H11001 ATP (b) ATP H11001 fructose 8n ADP H11001 fructose 6-phosphate 11. Coupling ATP Cleavage to an Unfavorable Reaction To explore the consequences of coupling ATP hydrolysis under physiological conditions to a thermodynamically unfavorable biochemical reaction, consider the hypothetical transformation X n Y, for which H9004GH11032H11034 H11005 20 kJ/mol. (a) What is the ratio [Y]/[X] at equilibrium? (b) Suppose X and Y participate in a sequence of reac- tions during which ATP is hydrolyzed to ADP and P i . The overall reaction is X H11001 ATP H11001 H 2 O 8n Y H11001 ADP H11001 P i Calculate [Y]/[X] for this reaction at equilibrium. Assume that the equilibrium concentrations of ATP, ADP, and P i are 1 M. (c) We know that [ATP], [ADP], and [P i ] are not 1 M un- der physiological conditions. Calculate [Y]/[X] for the ATP- coupled reaction when the values of [ATP], [ADP], and [P i ] are those found in rat myocytes (Table 13–5). 12. Calculations of H9004G at Physiological Concentrations Calculate the physiological H9004G (not H9004GH11032H11034) for the reaction Phosphocreatine H11001 ADP 8n creatine H11001 ATP at 25 H11034C, as it occurs in the cytosol of neurons, with phos- phocreatine at 4.7 mM, creatine at 1.0 mM, ADP at 0.73 mM, and ATP at 2.6 mM. Chapter 13 Principles of Bioenergetics520 13. Free Energy Required for ATP Synthesis under Physiological Conditions In the cytosol of rat hepato- cytes, the mass-action ratio, Q, is H5007 [A [ D A P T ] P [P ] i ] H5007 H11005 5.33 H11003 10 2 M H110021 Calculate the free energy required to synthesize ATP in a rat hepatocyte. 14. Daily ATP Utilization by Human Adults (a) A total of 30.5 kJ/mol of free energy is needed to synthesize ATP from ADP and P i when the reactants and products are at 1 M concentrations (standard state). Because the actual physiological concentrations of ATP, ADP, and P i are not 1 M, the free energy required to synthesize ATP un- der physiological conditions is different from H9004GH11032H11034. Calculate the free energy required to synthesize ATP in the human he- patocyte when the physiological concentrations of ATP, ADP, and P i are 3.5, 1.50, and 5.0 mM, respectively. (b) A 68 kg (150 lb) adult requires a caloric intake of 2,000 kcal (8,360 kJ) of food per day (24 h). The food is me- tabolized and the free energy is used to synthesize ATP, which then provides energy for the body’s daily chemical and me- chanical work. Assuming that the efficiency of converting food energy into ATP is 50%, calculate the weight of ATP used by a human adult in 24 h. What percentage of the body weight does this represent? (c) Although adults synthesize large amounts of ATP daily, their body weight, structure, and composition do not change significantly during this period. Explain this apparent contradiction. 15. Rates of Turnover of H9253 and H9252 Phosphates of ATP If a small amount of ATP labeled with radioactive phospho- rus in the terminal position, [H9253- 32 P]ATP, is added to a yeast extract, about half of the 32 P activity is found in P i within a few minutes, but the concentration of ATP remains un- changed. Explain. If the same experiment is carried out us- ing ATP labeled with 32 P in the central position, [H9252- 32 P]ATP, the 32 P does not appear in P i within such a short time. Why? 16. Cleavage of ATP to AMP and PP i during Metabo- lism The synthesis of the activated form of acetate (acetyl- CoA) is carried out in an ATP-dependent process: Acetate H11001 CoA H11001 ATP 8n acetyl-CoA H11001 AMP H11001 PP i (a) The H9004GH11032H11034 for the hydrolysis of acetyl-CoA to acetate and CoA is H1100232.2 kJ/mol and that for hydrolysis of ATP to AMP and PP i is H1100230.5 kJ/mol. Calculate H9004GH11032H11034 for the ATP- dependent synthesis of acetyl-CoA. (b) Almost all cells contain the enzyme inorganic py- rophosphatase, which catalyzes the hydrolysis of PP i to P i . What effect does the presence of this enzyme have on the synthesis of acetyl-CoA? Explain. 17. Energy for H H11545 Pumping The parietal cells of the stomach lining contain membrane “pumps” that transport hy- drogen ions from the cytosol of these cells (pH 7.0) into the stomach, contributing to the acidity of gastric juice (pH 1.0). Calculate the free energy required to transport 1 mol of hy- drogen ions through these pumps. (Hint: See Chapter 11.) Assume a temperature of 25 H11034C. 18. Standard Reduction Potentials The standard re- duction potential, EH11032H11034, of any redox pair is defined for the half-cell reaction: Oxidizing agent H11001 n electrons 8n reducing agent The EH11032H11034 values for the NAD H11001 /NADH and pyruvate/lactate con- jugate redox pairs are H110020.32 V and H110020.19 V, respectively. (a) Which conjugate pair has the greater tendency to lose electrons? Explain. (b) Which is the stronger oxidizing agent? Explain. (c) Beginning with 1 M concentrations of each reactant and product at pH 7, in which direction will the following re- action proceed? Pyruvate H11001 NADH H11001 H H11001 34 lactate H11001 NAD H11001 (d) What is the standard free-energy change (H9004GH11032H11034) at 25 H11034C for the conversion of pyruvate to lactate? (e) What is the equilibrium constant (KH11032 eq ) for this reaction? 19. Energy Span of the Respiratory Chain Electron transfer in the mitochondrial respiratory chain may be rep- resented by the net reaction equation NADH H11001 H H11001 H11001 H5007 1 2 H5007 O 2 34 H 2 O H11001 NAD H11001 (a) Calculate the value of H9004EH11032H11034 for the net reaction of mitochondrial electron transfer. Use EH11032H11034 values from Table 13–7. (b) Calculate H9004GH11032H11034 for this reaction. (c) How many ATP molecules can theoretically be gen- erated by this reaction if the free energy of ATP synthesis un- der cellular conditions is 52 kJ/mol? 20. Dependence of Electromotive Force on Concen- trations Calculate the electromotive force (in volts) regis- tered by an electrode immersed in a solution containing the following mixtures of NAD H11001 and NADH at pH 7.0 and 25 H11034C, with reference to a half-cell of EH11032H11034 0.00 V. (a) 1.0 mM NAD H11001 and 10 mM NADH (b) 1.0 mM NAD H11001 and 1.0 mM NADH (c) 10 mM NAD H11001 and 1.0 mM NADH 21. Electron Affinity of Compounds List the following substances in order of increasing tendency to accept elec- trons: (a) H9251-ketoglutarate H11001 CO 2 (yielding isocitrate); (b) ox- aloacetate; (c) O 2 ; (d) NADP H11001 . 22. Direction of Oxidation-Reduction Reactions Which of the following reactions would you expect to proceed in the direction shown, under standard conditions, assuming that the appropriate enzymes are present to catalyze them? (a) Malate H11001 NAD H11001 8n oxaloacetate H11001 NADH H11001 H H11001 (b) Acetoacetate H11001 NADH H11001 H H11001 8n H9252-hydroxybutyrate H11001 NAD H11001 (c) Pyruvate H11001 NADH H11001 H H11001 8n lactate H11001 NAD H11001 (d) Pyruvate H11001 H9252-hydroxybutyrate 8n lactate H11001 acetoacetate (e) Malate H11001 pyruvate 8n oxaloacetate H11001 lactate (f) Acetaldehyde H11001 succinate On ethanol H11001 fumarate chapter G lucose occupies a central position in the metabolism of plants, animals, and many microorganisms. It is relatively rich in potential energy, and thus a good fuel; the complete oxidation of glucose to carbon dioxide and water proceeds with a standard free-energy change of H110022,840 kJ/mol. By storing glucose as a high molecular weight polymer such as starch or glycogen, a cell can stockpile large quantities of hexose units while main- taining a relatively low cytosolic osmolarity. When en- ergy demands increase, glucose can be released from these intracellular storage polymers and used to pro- duce ATP either aerobically or anaerobically. Glucose is not only an excellent fuel, it is also a re- markably versatile precursor, capable of supplying a huge array of metabolic intermediates for biosynthetic reactions. A bacterium such as Escherichia coli can ob- tain from glucose the carbon skeletons for every amino acid, nucleotide, coenzyme, fatty acid, or other meta- bolic intermediate it needs for growth. A comprehen- sive study of the metabolic fates of glucose would en- compass hundreds or thousands of transformations. In animals and vascular plants, glucose has three major fates: it may be stored (as a polysaccharide or as su- crose); oxidized to a three-carbon compound (pyru- vate) via glycolysis to provide ATP and metabolic in- termediates; or oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose 5-phos- phate for nucleic acid synthesis and NADPH for reduc- tive biosynthetic processes (Fig. 14–1). Organisms that do not have access to glucose from other sources must make it. Photosynthetic organisms make glucose by first reducing atmospheric CO 2 to trioses, then converting the trioses to glucose. Non- photosynthetic cells make glucose from simpler three- and four-carbon precursors by the process of gluconeo- genesis, effectively reversing glycolysis in a pathway that uses many of the glycolytic enzymes. In this chapter we describe the individual reactions of glycolysis, gluconeogenesis, and the pentose phos- phate pathway and the functional significance of each pathway. We also describe the various fates of the pyruvate produced by glycolysis; they include the fer- mentations that are used by many organisms in anaer- obic niches to produce ATP and that are exploited in- dustrially as sources of ethanol, lactic acid, and other GLYCOLYSIS, GLUCONEOGENESIS, AND THE PENTOSE PHOSPHATE PATHWAY 14.1 Glycolysis 522 14.2 Feeder Pathways for Glycolysis 534 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation 538 14.4 Gluconeogenesis 543 14.5 Pentose Phosphate Pathway of Glucose Oxidation 549 The problem of alcoholic fermentation, of the origin and nature of that mysterious and apparently spontaneous change, which converted the insipid juice of the grape into stimulating wine, seems to have exerted a fascination over the minds of natural philosophers from the very earliest times. —Arthur Harden, Alcoholic Fermentation, 1923 14 521 O H OH O HO H CH 2 OCH 2 OH H P P 8885d_c14_521-559 2/6/04 3:43 PM Page 521 mac76 mac76:385_reb: commercially useful products. And we look at the path- ways that feed various sugars from mono-, di-, and poly- saccharides into the glycolytic pathway. The discussion of glucose metabolism continues in Chapter 15, where we describe the opposing anabolic and catabolic path- ways that connect glucose and glycogen, and use the processes of carbohydrate synthesis and degradation as examples of the many mechanisms by which organisms regulate metabolic pathways. 14.1 Glycolysis In glycolysis (from the Greek glykys, meaning “sweet,” and lysis, meaning “splitting”), a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is con- served in the form of ATP and NADH. Glycolysis was the first metabolic pathway to be elucidated and is prob- ably the best understood. From Eduard Buchner’s dis- covery in 1897 of fermentation in broken extracts of yeast cells until the elucidation of the whole pathway in yeast (by Otto Warburg and Hans von Euler-Chelpin) and in muscle (by Gustav Embden and Otto Meyerhof) in the 1930s, the reactions of glycolysis in extracts of yeast and muscle were a major focus of biochemical re- search. The philosophical shift that accompanied these discoveries was announced by Jacques Loeb in 1906: Through the discovery of Buchner, Biology was relieved of another fragment of mysticism. The splitting up of sugar into CO 2 and alcohol is no more the effect of a “vital principle” than the splitting up of cane sugar by invertase. The history of this problem is instructive, as it warns us against considering problems as beyond our reach because they have not yet found their solution. The development of methods of enzyme purifica- tion, the discovery and recognition of the importance of coenzymes such as NAD, and the discovery of the piv- otal metabolic role of ATP and other phosphorylated compounds all came out of studies of glycolysis. The gly- colytic enzymes of many species have long since been purified and thoroughly studied. Glycolysis is an almost universal central pathway of glucose catabolism, the pathway with the largest flux of carbon in most cells. The glycolytic breakdown of glu- cose is the sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal medulla, brain, and sperm, for example). Some plant tis- sues that are modified to store starch (such as potato tubers) and some aquatic plants (watercress, for ex- ample) derive most of their energy from glycolysis; many anaerobic microorganisms are entirely dependent on glycolysis. Fermentation is a general term for the anaerobic degradation of glucose or other organic nutrients to ob- tain energy, conserved as ATP. Because living organisms first arose in an atmosphere without oxygen, anaerobic breakdown of glucose is probably the most ancient bio- logical mechanism for obtaining energy from organic fuel molecules. In the course of evolution, the chemistry of this reaction sequence has been completely con- served; the glycolytic enzymes of vertebrates are closely similar, in amino acid sequence and three-dimensional structure, to their homologs in yeast and spinach. Gly- colysis differs among species only in the details of its regulation and in the subsequent metabolic fate of the pyruvate formed. The thermodynamic principles and the types of regulatory mechanisms that govern glycolysis are common to all pathways of cell me- tabolism. A study of glycolysis can therefore serve as a model for many aspects of the pathways discussed throughout this book. Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway522 Ribose 5-phosphate Pyruvate Glycogen, starch, sucrose oxidation via pentose phosphate pathway oxidation via glycolysis Glucose storage FIGURE 14–1 Major pathways of glucose utilization. Although not the only possible fates for glucose, these three pathways are the most significant in terms of the amount of glucose that flows through them in most cells. Hans von Euler-Chelpin, 1873–1964 Gustav Embden, 1874–1933 Otto Meyerhof, 1884–1951 8885d_c14_521-559 2/6/04 3:43 PM Page 522 mac76 mac76:385_reb: Before examining each step of the pathway in some detail, we take a look at glycolysis as a whole. An Overview: Glycolysis Has Two Phases The breakdown of the six-carbon glucose into two mol- ecules of the three-carbon pyruvate occurs in ten steps, the first five of which constitute the preparatory phase (Fig. 14–2a). In these reactions, glucose is first phos- phorylated at the hydroxyl group on C-6 (step 1 ). The D-glucose 6-phosphate thus formed is converted to D- fructose 6-phosphate (step 2 ), which is again phos- phorylated, this time at C-1, to yield D-fructose 1,6- bisphosphate (step 3 ). For both phosphorylations, ATP is the phosphoryl group donor. As all sugar derivatives in glycolysis are the D isomers, we will usually omit the D designation except when emphasizing stereochemistry. Fructose 1,6-bisphosphate is split to yield two three-carbon molecules, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (step 4 ); this is the “lysis” step that gives the pathway its name. The dihy- droxyacetone phosphate is isomerized to a second mol- ecule of glyceraldehyde 3-phosphate (step 5 ), ending the first phase of glycolysis. From a chemical perspec- tive, the isomerization in step 2 is critical for setting up the phosphorylation and COC bond cleavage reac- tions in steps 3 and 4 , as detailed later. Note that two molecules of ATP are invested before the cleavage of glucose into two three-carbon pieces; later there will be a good return on this investment. To summarize: in the preparatory phase of glycolysis the energy of ATP is invested, raising the free-energy content of the inter- mediates, and the carbon chains of all the metabolized hexoses are converted into a common product, glyceraldehyde 3-phosphate. The energy gain comes in the payoff phase of gly- colysis (Fig. 14–2b). Each molecule of glyceraldehyde 3-phosphate is oxidized and phosphorylated by inor- ganic phosphate (not by ATP) to form 1,3-bisphospho- glycerate (step 6 ). Energy is then released as the two molecules of 1,3-bisphosphoglycerate are converted to two molecules of pyruvate (steps 7 through 10). Much of this energy is conserved by the coupled phosphory- lation of four molecules of ADP to ATP. The net yield is two molecules of ATP per molecule of glucose used, be- cause two molecules of ATP were invested in the preparatory phase. Energy is also conserved in the pay- off phase in the formation of two molecules of NADH per molecule of glucose. In the sequential reactions of glycolysis, three types of chemical transformations are particularly noteworthy: (1) degradation of the carbon skeleton of glucose to yield pyruvate, (2) phosphorylation of ADP to ATP by high-energy phosphate compounds formed during glycolysis, and (3) transfer of a hydride ion to NAD H11001 , forming NADH. Fates of Pyruvate With the exception of some interest- ing variations in the bacterial realm, the pyruvate formed by glycolysis is further metabolized via one of three catabolic routes. In aerobic organisms or tissues, under aerobic conditions, glycolysis is only the first stage in the complete degradation of glucose (Fig. 14–3). Pyru- vate is oxidized, with loss of its carboxyl group as CO 2 , to yield the acetyl group of acetyl-coenzyme A; the acetyl group is then oxidized completely to CO 2 by the citric acid cycle (Chapter 16). The electrons from these oxidations are passed to O 2 through a chain of carriers in the mitochondrion, to form H 2 O. The energy from the electron-transfer reactions drives the synthesis of ATP in the mitochondrion (Chapter 19). The second route for pyruvate is its reduction to lactate via lactic acid fermentation. When vigorously contracting skeletal muscle must function under low- oxygen conditions (hypoxia), NADH cannot be reoxi- dized to NAD H11001 , but NAD H11001 is required as an electron ac- ceptor for the further oxidation of pyruvate. Under these conditions pyruvate is reduced to lactate, accepting electrons from NADH and thereby regenerating the NAD H11001 necessary for glycolysis to continue. Certain tis- sues and cell types (retina and erythrocytes, for exam- ple) convert glucose to lactate even under aerobic con- ditions, and lactate is also the product of glycolysis under anaerobic conditions in some microorganisms (Fig. 14–3). The third major route of pyruvate catabolism leads to ethanol. In some plant tissues and in certain inver- tebrates, protists, and microorganisms such as brewer’s yeast, pyruvate is converted under hypoxic or anaero- bic conditions into ethanol and CO 2 , a process called ethanol (alcohol) fermentation (Fig. 14–3). The oxidation of pyruvate is an important catabolic process, but pyruvate has anabolic fates as well. It can, for example, provide the carbon skeleton for the syn- thesis of the amino acid alanine. We return to these an- abolic reactions of pyruvate in later chapters. ATP Formation Coupled to Glycolysis During glycolysis some of the energy of the glucose molecule is conserved in ATP, while much remains in the product, pyruvate. The overall equation for glycolysis is Glucose H11001 2NAD H11001 H11001 2ADP H11001 2P i 88n 2 pyruvate H11001 2NADH H11001 2H H11001 H11001 2ATP H11001 2H 2 O (14–1) For each molecule of glucose degraded to pyruvate, two molecules of ATP are generated from ADP and P i . We can now resolve the equation of glycolysis into two processes—the conversion of glucose to pyruvate, which is exergonic: Glucose H11001 2NAD H11001 88n 2 pyruvate H11001 2NADH H11001 2H H11001 (14–2) H9004G 1 H11032H11034 H11005 H11002146 kJ/mol 14.1 Glycolysis 523 8885d_c14_523 2/9/04 7:01 AM Page 523 mac76 mac76:385_reb: Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway524 O cleavage of 6-carbon sugar phosphate to two 3-carbon sugar phosphates 5 first priming reaction 2 1 H H OH HH HO Dihydroxyacetone phosphate Glucose Glucose 6-phosphate second ATP- forming reaction (substrate-level phosphorylation) 10 ADP Fructose 6-phosphate Fructose 1,6-bisphosphate 3-Phosphoglycerate (2) CH 2 OH HO O H H H OH OH HH HO O OH H O H HO H P O CH 2 OH CH 2 OH O H OHO H CH 2 HO H C CH 2 OCH 2 OH H CH 2 OH CH C O O CH 2 O Payoff phase Oxidative conversion of glyceraldehyde 3-phosphate to pyruvate and the coupled formation of ATP and NADH C OHCH 2 O Glyceraldehyde 3-phosphate ADP second priming reaction 3 2P i 2ADP Glyceraldehyde 3-phosphate (2) 2NAD H11001 Pyruvate (2) 2ADP 1,3-Bisphosphoglycerate (2) CH 2 CH O 2H 2 O 2-Phosphoglycerate (2) Phosphoenolpyruvate (2) first ATP- forming reaction (substrate-level phosphorylation) 6 7 9 8 4 O C O Preparatory phase Phosphorylation of glucose and its conversion to glyceraldehyde 3-phosphate (a) (b) H11001 oxidation and phosphorylation 2 H11001 H H11001 ATP ATP P P P P P P P OH C O H CH 2 CHOP OH CCH 2 CHOP OH CH 2 CH OH P CH 3 C O H11002 O P CH 2 C 1 2 3 4 5 6 NADH2 ATP 2ATP OH OH C O H O O C O H11002 O O H11002 O O H11002 O 1 2 3 4 5 Hexokinase Phosphohexose isomerase Phospho- fructokinase-1 Aldolase Triose phosphate isomerase 6 7 8 9 10 Glyceraldehyde 3-phosphate dehydrogenase Phospho- glycerate kinase Phospho- glycerate mutase Enolase Pyruvate kinase FIGURE 14–2 The two phases of glycolysis. For each molecule of glu- cose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycol- ysis. For each glucose molecule, two ATP are consumed in the prepara- tory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. The numbered reaction steps are catalyzed by the enzymes listed on the right, and also correspond to the numbered headings in the text discussion. Keep in mind that each phosphoryl group, represented here as P , has two negative charges (OPO 3 2H11002 ). 8885d_c14_521-559 2/6/04 3:43 PM Page 524 mac76 mac76:385_reb: and the formation of ATP from ADP and P i , which is endergonic: 2ADP H11001 2P i 88n 2ATP H11001 2H 2 O (14–3) H9004G 2 H11032H11034 H11005 2(30.5 kJ/mol) H11005 61.0 kJ/mol The sum of Equations 14–2 and 14–3 gives the overall standard free-energy change of glycolysis, H9004G s H11032H11034: H9004G s H11032H11034 H11005 H9004G 1 H11032H11034 H11001 H9004G 2 H11032H11034 H11005 H11002146 kJ/mol H11001 61.0 kJ/mol H11005H1100285 kJ/mol Under standard conditions and in the cell, glycolysis is an essentially irreversible process, driven to completion by a large net decrease in free energy. At the actual in- tracellular concentrations of ATP, ADP, and P i (see Box 13–1) and of glucose and pyruvate, the energy released in glycolysis (with pyruvate as the end product) is re- covered as ATP with an efficiency of more than 60%. Energy Remaining in Pyruvate Glycolysis releases only a small fraction of the total available energy of the glu- cose molecule; the two molecules of pyruvate formed by glycolysis still contain most of the chemical poten- tial energy of glucose, energy that can be extracted by oxidative reactions in the citric acid cycle (Chapter 16) and oxidative phosphorylation (Chapter 19). Importance of Phosphorylated Intermediates Each of the nine glycolytic intermediates between glucose and pyru- vate is phosphorylated (Fig. 14–2). The phosphoryl groups appear to have three functions. 1. Because the plasma membrane generally lacks transporters for phosphorylated sugars, the phos- phorylated glycolytic intermediates cannot leave the cell. After the initial phosphorylation, no fur- ther energy is necessary to retain phosphorylated intermediates in the cell, despite the large differ- ence in their intracellular and extracellular con- centrations. 2. Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. Energy released in the breakage of phosphoanhy- dride bonds (such as those in ATP) is partially conserved in the formation of phosphate esters such as glucose 6-phosphate. High-energy phos- phate compounds formed in glycolysis (1,3-bisphos- phoglycerate and phosphoenolpyruvate) donate phosphoryl groups to ADP to form ATP. 3. Binding energy resulting from the binding of phos- phate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions (Chapter 6). The phos- phate groups of ADP, ATP, and the glycolytic in- termediates form complexes with Mg 2H11001 , and the substrate binding sites of many glycolytic enzymes are specific for these Mg 2H11001 complexes. Most gly- colytic enzymes require Mg 2H11001 for activity. The Preparatory Phase of Glycolysis Requires ATP In the preparatory phase of glycolysis, two molecules of ATP are invested and the hexose chain is cleaved into two triose phosphates. The realization that phosphory- lated hexoses were intermediates in glycolysis came slowly and serendipitously. In 1906, Arthur Harden and William Young tested their hypothesis that inhibitors of proteolytic enzymes would stabilize the glucose- fermenting enzymes in yeast extract. They added blood serum (known to contain inhibitors of proteolytic en- zymes) to yeast extracts and observed the predicted stimulation of glucose metabolism. However, in a con- trol experiment intended to show that boiling the serum destroyed the stimulatory activity, they discovered that boiled serum was just as effective at stimulating glycol- ysis. Careful examination and testing of the contents of 14.1 Glycolysis 525 Glucose 2 Pyruvate 2 Acetyl-CoA 4CO 2 H11001 4H 2 O 2 Ethanol H11001 2CO 2 2 Lactate glycolysis (10 successive reactions) aerobic conditions 2CO 2 citric acid cycle Fermentation to lactate in vigor- ously contracting muscle, in erythro- cytes, in some other cells, and in some micro- organisms anaerobic conditions hypoxic or anaerobic conditions Animal, plant, and many microbial cells under aerobic conditions Fermentation to ethanol in yeast FIGURE 14–3 Three possible catabolic fates of the pyruvate formed in glycolysis. Pyruvate also serves as a precursor in many anabolic re- actions, not shown here. Arthur Harden, 1865–1940 William Young, 1878–1942 8885d_c14_521-559 2/6/04 3:43 PM Page 525 mac76 mac76:385_reb: the boiled serum revealed that inorganic phosphate was responsible for the stimulation. Harden and Young soon discovered that glucose added to their yeast extract was converted to a hexose bisphosphate (the “Harden- Young ester,” eventually identified as fructose 1,6- bisphosphate). This was the beginning of a long series of investigations on the role of organic esters of phos- phate in biochemistry, which has led to our current un- derstanding of the central role of phosphoryl group transfer in biology. 1 Phosphorylation of Glucose In the first step of glycol- ysis, glucose is activated for subsequent reactions by its phosphorylation at C-6 to yield glucose 6-phosphate, with ATP as the phosphoryl donor: This reaction, which is irreversible under intracel- lular conditions, is catalyzed by hexokinase. Recall that kinases are enzymes that catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor nu- cleophile (see Fig. 13–10). Kinases are a subclass of transferases (see Table 6–3). The acceptor in the case of hexokinase is a hexose, normally D-glucose, although hexokinase also catalyzes the phosphorylation of other common hexoses, such as D-fructose and D-mannose. Hexokinase, like many other kinases, requires Mg 2H11001 for its activity, because the true substrate of the enzyme is not ATP 4H11002 but the MgATP 2H11002 complex (see Fig. 13–2). Mg 2H11001 shields the negative charges of the phosphoryl groups in ATP, making the terminal phosphorus atom an easier target for nucleophilic attack by an OOH of glu- cose. Hexokinase undergoes a profound change in shape, an induced fit, when it binds glucose; two do- mains of the protein move about 8 ? closer to each other when ATP binds (see Fig. 6–22). This movement brings bound ATP closer to a molecule of glucose also bound to the enzyme and blocks the access of water (from the solvent), which might otherwise enter the active site and attack (hydrolyze) the phosphoanhydride bonds of ATP. Like the other nine enzymes of glycolysis, hexo- kinase is a soluble, cytosolic protein. Hexokinase is present in all cells of all organisms. Hepatocytes also contain a form of hexokinase called hexokinase IV or glucokinase, which differs from other forms of hexokinase in kinetic and regulatory proper- ties (see Box 15–2). Two enzymes that catalyze the O OPO 3 2H11002 H OH HO H H H OHH CH 2 OH O O H OH HO H H H OHH CH 2 OH O OH ATP ADP Glucose Glucose 6-phosphate H11005H1100216.7 kJ/molDGH11032H11034 hexokinase Mg 2H11001 5 6 41 2 3 same reaction but are encoded in different genes are called isozymes. 2 Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate The enzyme phosphohexose isomerase (phospho- glucose isomerase) catalyzes the reversible isomer- ization of glucose 6-phosphate, an aldose, to fructose 6-phosphate, a ketose: The mechanism for this reaction is shown in Figure 14–4. The reaction proceeds readily in either direction, as might be expected from the relatively small change in standard free energy. This isomerization has a criti- cal role in the overall chemistry of the glycolytic path- way, as the rearrangement of the carbonyl and hydroxyl groups at C-1 and C-2 is a necessary prelude to the next two steps. The phosphorylation that occurs in the next reaction (step 3 ) requires that the group at C-1 first be converted from a carbonyl to an alcohol, and in the subsequent reaction (step 4 ) cleavage of the bond be- tween C-3 and C-4 requires a carbonyl group at C-2 (p. 485). 3 Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate In the second of the two priming reactions of glycolysis, phosphofructokinase-1 (PFK-1) cat- alyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphos- phate: OPO 3 2H11002 ATP ADP phosphofructokinase-1 (PFK-1) Mg 2H11001 O HO H H OH H CH 2 OH OH Fructose 6-phosphate Fructose 1,6-bisphosphate H11005H1100214.2 kJ/molDGH11032H11034 O H H OH H CH 2 OH HO 6 5 43 6 1 5 43 2 1 2 CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 O HO OH H H OH H CH 2 OH H OH HO H H H OHH OH Glucose 6-phosphate Fructose 6-phosphate H11005 1.7 kJ/molDGH11032H11034 Mg 2H11001 phosphohexose isomerase 4 2 1 3 6 5 4 3 2 1O 6 5 CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway526 8885d_c14_521-559 2/6/04 3:43 PM Page 526 mac76 mac76:385_reb: This enzyme is called PFK-1 to distinguish it from a sec- ond enzyme (PFK-2) that catalyzes the formation of fructose 2,6-bisphosphate from fructose 6-phosphate in a separate pathway. The PFK-1 reaction is essentially irreversible under cellular conditions, and it is the first “committed” step in the glycolytic pathway; glucose 6-phosphate and fructose 6-phosphate have other pos- sible fates, but fructose 1,6-bisphosphate is targeted for glycolysis. Some bacteria and protists and perhaps all plants have a phosphofructokinase that uses pyrophosphate (PP i ), not ATP, as the phosphoryl group donor in the synthesis of fructose 1,6-bisphosphate: Mg 2H11001 Fructose 6-phosphate H11001 PP i 88n fructose 1,6-bisphosphate H11001 P i H9004GH11032H11034 H11005 H1100214 kJ/mol Phosphofructokinase-1 is a regulatory enzyme (Chapter 6), one of the most complex known. It is the major point of regulation in glycolysis. The activity of PFK-1 is increased whenever the cell’s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), are in excess. The en- zyme is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. In some organisms, fructose 2,6-bisphosphate (not to be confused with the PFK-1 reaction product, fructose 1,6- bisphosphate) is a potent allosteric activator of PFK-1. The regulation of this step in glycolysis is discussed in greater detail in Chapter 15. 4 Cleavage of Fructose 1,6-Bisphosphate The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase, catalyzes a reversible aldol condensa- tion (p. 485). Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, glyceraldehyde 3-phosphate, an aldose, and dihydroxyacetone phosphate, a ketose: There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14–5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2; the Zn 2H11001 polarizes the carbonyl group CHOH Glyceraldehyde 3-phosphate H11005 23.8 kJ/molDGH11032H11034 H11001 aldolase C O H OH C CH 2 O Dihydroxyacetone phosphate O H H OH H OH Fructose 1,6-bisphosphate HO 1 (1) 2 (2) 5 (5) 4 (4) 3 (3) 6 (6) CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 14.1 Glycolysis 527 HOH HO HH 6 CH 2 OPO 3 2– 6 CH 2 OPO 3 2– HOH OH Glucose 6-phosphate Phosphohexose isomerase binding and ring opening O HOH H 1 CH 2 OH OH H OH Fructose 6-phosphate : OH B HO 3 CH 2 C 1 C OH H + H H 4 COH H 5 COH 6 CH 2 OPO 3 2– :B HOCH C CO H OHH HCOH HCOH cis-Enediol intermediate OHH BH HOCH C C OH H + HCOH HCOH CH 2 OPO 3 2– CH 2 OPO 3 2– 1 ring closing and dissociation 4 2 3 1 2 2 3 34 4 5 5 O MECHANISM FIGURE 14–4 The phosphohexose isomerase reaction. The ring opening and closing reactions (steps 1 and 4 ) are catalyzed by an active-site His residue, by mechanisms omitted here for simplicity. The movement of the proton between C-2 and C-1 (steps 2 and 3 ) is base-catalyzed by an active-site Glu residue (shown as B:). The proton (pink) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and the nearby hydroxyl group. After its transfer from C-2 to the active-site Glu residue, the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step 2 is not necessarily the same one that is added to C-1 in step 3 . (The additional exchange of protons (yellow and blue) between the hydroxyl groups and solvent is shown for completeness. The hydroxyl groups are weak acids and can exchange protons with the surrounding water whether the isomerization reaction is underway or not.) Phosphohexose Isomerase Mechanism 8885d_c14_527 2/9/04 7:02 AM Page 527 mac76 mac76:385_reb: and stabilizes the enolate intermediate created in the COC bond cleavage step. Although the aldolase reaction has a strongly posi- tive standard free-energy change in the direction of fruc- tose 1,6-bisphosphate cleavage, at the lower concentra- tions of reactants present in cells, the actual free-energy change is small and the aldolase reaction is readily re- versible. We shall see later that aldolase acts in the re- verse direction during the process of gluconeogenesis (see Fig. 14–16). 5 Interconversion of the Triose Phosphates Only one of the two triose phosphates formed by aldolase, glyceralde- hyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis. The other product, dihy- droxyacetone phosphate, is rapidly and reversibly Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway528 Aldolase binding and ring opening : : H N H Lys HOCH C OH B :B A H + HCOH CH 2 OPO 3 2– 1 CH 2 OPO 3 2– CH 2 OPO 3 2– CH 2 OPO 3 2– Fructose 1,6-bisphosphate : :N + H H HOCH C OH H + B :B A H 2 O HC HO CH 2 OPO 3 2– 2 3 CH 2 OPO 3 2– HC HO : H N H Lys H :B A : N + H Lys HOCH C B BH HCOH CH 2 OPO 3 2– CH 2 OPO 3 2– CH 2 OPO 3 2– : A HC HO N H Lys C C BH O H H Enamine intermediate CH 2 OPO 3 2– HB : : A In steps 1 and 2 an amine is converted to a Schiff base (imine). Proton exchange with solution restores enzyme. first product released second product released N + H Lys C C H HHO B CH 2 OPO 3 2– HB : : A H 2 O 5 : B Schiff base is hydrolyzed in reverse of Schiff base formation. OH C HCOH Glyceraldehyde 3-phosphate CH 2 OPO 3 CO 2– Dihydroxy- acetone phosphate CH 2 OH 4 Protonated Schiff base Protonated Schiff base HOH H HO H OH O MECHANISM FIGURE 14–5 The class I aldolase reaction. The reac- tion shown here is the reverse of an aldol condensation. Note that cleavage between C-3 and C-4 depends on the presence of the car- bonyl group at C-2. 1 and 2 The carbonyl reacts with an active-site Lys residue to form an imine, which stabilizes the carbanion generated by the bond cleavage—an imine delocalizes electrons even better than does a carbonyl. 3 Bond cleavage releases glyceraldeyde 3-phosphate as the first product. 4 The resulting enamine covalently linked to the enzyme is isomerized to a protonated Schiff base, and 5 hydrolysis of the Schiff base generates dihydroxyacetone phosphate as the sec- ond product. A and B represent amino acid residues that serve as general acid (A) or base (B). 8885d_c14_528 2/9/04 7:02 AM Page 528 mac76 mac76:385_reb: converted to glyceraldehyde 3-phosphate by the fifth enzyme of the sequence, triose phosphate isomerase: The reaction mechanism is similar to the reaction pro- moted by phosphohexose isomerase in step 2 of gly- colysis (Fig. 14–4). After the triose phosphate isomerase reaction, C-1, C-2, and C-3 of the starting glucose are chemically indistinguishable from C-6, C-5, and C-4, re- spectively (Fig. 14–6), setting up the efficient metabo- lism of the entire six-carbon glucose molecule. This reaction completes the preparatory phase of glycolysis. The hexose molecule has been phosphory- lated at C-1 and C-6 and then cleaved to form two mol- ecules of glyceraldehyde 3-phosphate. The Payoff Phase of Glycolysis Yields ATP and NADH The payoff phase of glycolysis (Fig. 14–2b) includes the energy-conserving phosphorylation steps in which some of the free energy of the glucose molecule is conserved in the form of ATP. Remember that one molecule of glu- cose yields two molecules of glyceraldehyde 3-phos- phate; both halves of the glucose molecule follow the same pathway in the second phase of glycolysis. The conversion of two molecules of glyceraldehyde 3-phos- phate to two molecules of pyruvate is accompanied by the formation of four molecules of ATP from ADP. How- ever, the net yield of ATP per molecule of glucose de- graded is only two, because two ATP were invested in the preparatory phase of glycolysis to phosphorylate the two ends of the hexose molecule. 6 Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphos- phoglycerate The first step in the payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bis- phosphoglycerate, catalyzed by glyceraldehyde 3- phosphate dehydrogenase: 14.1 Glycolysis 529 O OH OH CH 2 C CH 2 C C C P CH 2 O O O H OH OHH C C CH 2 OH P O O OCH 2 P H C 6 1 2 3 4 5 (b) Subsequent reactions of glycolysis Dihydroxyacetone phosphate 4 or 3 5 or 2 6 or 1 4 5 6 Derived from glucose carbons Fructose 1,6-bisphosphate triose phosphate isomerase HHO CH 2 C C O O 1 2 3 H P 1 2 3 Derived from glucose carbon Derived from glucose carbon Glyceraldehyde 3-phosphate (a) P D-Glyceraldehyde 3-phosphate aldolase H H FIGURE 14–6 Fate of the glucose carbons in the formation of glyc- eraldehyde 3-phosphate. (a) The origin of the carbons in the two three- carbon products of the aldolase and triose phosphate isomerase re- actions. The end product of the two reactions is glyceraldehyde 3-phosphate (two molecules). (b) Each carbon of glyceraldehyde 3-phosphate is derived from either of two specific carbons of glucose. Note that the numbering of the carbon atoms of glyceraldehyde 3-phosphate differs from that of the glucose from which it is derived. In glyceraldehyde 3-phosphate, the most complex functional group (the carbonyl) is specified as C-1. This numbering change is important for in- terpreting experiments with glucose in which a single carbon is labeled with a radioisotope. (See Problems 3 and 5 at the end of this chapter.) HCOH 1,3-Bisphosphoglycerate H11005 6.3 kJ/molDGH11032H11034 NAD H11001 H H11001 glyceraldehyde 3-phosphate dehydrogenase C OPO 3 O H Inorganic phosphate O 2H11002 O PHOH11001 H11002 C OO H11002 O PO H11001 H11002 Glyceraldehyde 3-phosphate CH 2 H11002 OPO 3 2H11002 CH 2 O NADH O HCOH H11005 7.5 kJ/molDGH11032H11034 HCOH Glyceraldehyde 3-phosphate triose phosphate isomerase CCH 2 O H OH C O Dihydroxyacetone phosphate CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 8885d_c14_529 2/9/04 7:03 AM Page 529 mac76 mac76:385_reb: This is the first of the two energy-conserving reactions of glycolysis that eventually lead to the formation of ATP. The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid. This type of an- hydride, called an acyl phosphate, has a very high stan- dard free energy of hydrolysis (H9004GH11032H11034 H11005 H1100249.3 kJ/mol; see Fig. 13–4, Table 13–6). Much of the free energy of oxidation of the aldehyde group of glyceraldehyde 3- phosphate is conserved by formation of the acyl phos- phate group at C-1 of 1,3-bisphosphoglycerate. The acceptor of hydrogen in the glyceraldehyde 3- phosphate dehydrogenase reaction is NAD H11001 (see Fig. 13–15), bound to a Rossmann fold as shown in Figure 13–16. The reduction of NAD H11001 proceeds by the enzy- matic transfer of a hydride ion (:H H11002 ) from the aldehyde group of glyceraldehyde 3-phosphate to the nicoti- namide ring of NAD H11001 , yielding the reduced coenzyme NADH. The other hydrogen atom of the substrate mol- ecule is released to the solution as H H11001 . Glyceraldehyde 3-phosphate is covalently bound to the dehydrogenase during the reaction (Fig. 14–7). The aldehyde group of glyceraldehyde 3-phosphate reacts with the OSH group of an essential Cys residue in the active site, in a reaction analogous to the formation of a hemiacetal (see Fig. 7–5), in this case producing a thio- hemiacetal. Reaction of the essential Cys residue with a heavy metal such as Hg 2H11001 irreversibly inhibits the enzyme. Because cells maintain only limited amounts of NAD H11001 , glycolysis would soon come to a halt if the NADH formed in this step of glycolysis were not continuously reoxidized. The reactions in which NAD H11001 is regenerated anaerobically are described in detail in Section 14.3, in our discussion of the alternative fates of pyruvate. Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway530 C HCOH O 1,3-Bisphosphoglycerate 2 1 NAD + S H Cys : N NH His formation of thiohemiacetal intermediate C HCOH HO CH 2 OPO 3 2– NAD + S Cys C HCOH HO – CH 2 OPO 3 2– NAD + S H Cys : N NH His 3 oxidation to thioester intermediate NADH S Cys C HCOH O CH 2 OPO 3 2– NAD + S Cys C HCOH O CH 2 OPO 3 2– CH 2 OPO 3 2– OPO 3 2– O – – O POH O Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate formation of enzyme- substrate complex 4 NADH exchanged for NAD + ; attack on thioester by P i NAD + NADH P i 5 release of product H N NH His + H N NH His + H N NH His + MECHANISM FIGURE 14–7 The glyceraldehyde 3-phosphate dehy- drogenase reaction. After 1 formation of the enzyme-substrate com- plex, 2 a covalent thiohemiacetal linkage forms between the sub- strate and the OSH group of a Cys residue—facilitated by acid-base catalysis with a neighboring base catalyst, probably a His residue. 3 This enzyme-substrate intermediate is oxidized by NAD H11001 bound to the active site, forming a covalent acyl-enzyme intermediate, a thioester. 4 The newly formed NADH leaves the active site and is replaced by another NAD H11001 molecule. The bond between the acyl group and the thiol group of the enzyme has a very high standard free energy of hydrolysis. 5 This bond undergoes phosphorolysis (attack by P i ), releasing the acyl phosphate product, 1,3-bisphosphoglycerate. Formation of this product conserves much of the free energy liberated during oxidation of the aldehyde group of glyceraldehyde 3-phosphate. 8885d_c14_530 2/9/04 7:03 AM Page 530 mac76 mac76:385_reb: 7 Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate: Notice that [H H11001 ] is not included in Q. In biochemical cal- culations, [H H11001 ] is assumed to be a constant (10 H110027 M), and this constant is included in the definition of H9004GH11032H11034 (p. 491). When the mass-action ratio is less than 1.0, its nat- ural logarithm has a negative sign. Step 7 , by consum- ing the product of step 6 (1,3-bisphosphoglycerate), keeps [1,3-bisphosphoglycerate] relatively low in the steady state and thereby keeps Q for the overall energy- coupling process small. When Q is small, the contribution of ln Q can make H9004G strongly negative. This is simply another way of showing how the two reactions, steps 6 and 7 , are coupled through a common intermediate. The outcome of these coupled reactions, both re- versible under cellular conditions, is that the energy re- leased on oxidation of an aldehyde to a carboxylate group is conserved by the coupled formation of ATP from ADP and P i . The formation of ATP by phosphoryl group transfer from a substrate such as 1,3-bisphos- phoglycerate is referred to as a substrate-level phosphorylation, to distinguish this mechanism from respiration-linked phosphorylation. Substrate-level phosphorylations involve soluble enzymes and chemical intermediates (1,3-bisphosphoglycerate in this case). Respiration-linked phosphorylations, on the other hand, involve membrane-bound enzymes and transmembrane gradients of protons (Chapter 19). 8 Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate The enzyme phosphoglycerate mutase catalyzes a re- versible shift of the phosphoryl group between C-2 and C-3 of glycerate; Mg 2H11001 is essential for this reaction: The reaction occurs in two steps (Fig. 14–8). A phos- phoryl group initially attached to a His residue of the mutase is transferred to the hydroxyl group at C-2 of 3- phosphoglycerate, forming 2,3-bisphosphoglycerate (2,3-BPG). The phosphoryl group at C-3 of 2,3-BPG is then transferred to the same His residue, producing 2- phosphoglycerate and regenerating the phosphorylated enzyme. Phosphoglycerate mutase is initially phospho- rylated by phosphoryl transfer from 2,3-BPG, which is required in small quantities to initiate the catalytic cy- cle and is continuously regenerated by that cycle. Al- though in most cells 2,3-BPG is present in only trace amounts, it is a major component (~5 mM) of erythro- cytes, where it regulates the affinity of hemoglobin for 14.1 Glycolysis 531 O P O O P H11002 H11002 H11002 H11002 H11002 H11002 H11002 H11001 H11001 O OPO 3 2 O C HCOH CH 2 H11001 OO O P AdenineRib Mg 2 phosphoglycerate kinase O O OPO 3 2 C HCOH CH 2 AdenineRib O ATP 1,3-Bisphosphoglycerate 3-Phosphoglycerate ADP P P O P H9004GH11032H11034 H11005 H1100218.5 kJ/mol Notice that phosphoglycerate kinase is named for the reverse reaction. Like all enzymes, it catalyzes the re- action in both directions. This enzyme acts in the di- rection suggested by its name during gluconeogenesis (see Fig. 14–16) and during photosynthetic CO 2 assim- ilation (see Fig. 20–4). Steps 6 and 7 of glycolysis together constitute an energy-coupling process in which 1,3-bisphosphoglyc- erate is the common intermediate; it is formed in the first reaction (which would be endergonic in isolation), and its acyl phosphate group is transferred to ADP in the second reaction (which is strongly exergonic). The sum of these two reactions is Glyceraldehyde 3-phosphate H11001 ADP H11001 P i H11001 NAD H11001 3-phosphoglycerate H11001 ATP H11001 NADH H11001 H H11001 H9004GH11032H11034 H11005 H1100212.5 kJ/mol Thus the overall reaction is exergonic. Recall from Chapter 13 that the actual free-energy change, H9004G, is determined by the standard free-energy change, H9004GH11032H11034, and the mass-action ratio, Q, which is the ratio [products]/[reactants] (see Eqn 13–3). For step 6 H9004G H11005H9004GH11032H11034 H11001 RT ln Q H11005H9004GH11032H11034 H11001 RT ln [1,3-bisphosphoglycerate][NADH] H5007H5007H5007H5007H5007 [glyceraldehyde 3-phosphate][P i ][NAD H11001 ] z y H11005 4.4 kJ/molH9004GH11032° O H11002 H11002 C HC CH 2 O Mg 2H11001 phosphoglycerate mutase O O C HC CH 2 3-Phosphoglycerate 2-Phosphoglycerate O OH OH O PO 3 2H11002 O PO 3 2H11002 O 8885d_c14_531 2/9/04 7:03 AM Page 531 mac76 mac76:385_reb: oxygen (see Fig. 5–17; note that in the context of he- moglobin regulation, 2,3-bisphosphoglycerate is usually abbreviated as simply BPG). 9 Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate In the second glycolytic reaction that generates a com- pound with high phosphoryl group transfer potential, enolase promotes reversible removal of a molecule of water from 2-phosphoglycerate to yield phospho- enolpyruvate (PEP): The mechanism of the enolase reaction is presented in Figure 6–23. Despite the relatively small standard free- energy change of this reaction, there is a very large difference in the standard free energy of hydrolysis of the phosphoryl groups of the reactant and product: H1100217.6 kJ/mol for 2-phosphoglycerate (a low-energy phos- phate ester) and H1100261.9 kJ/mol for phosphoenolpyruvate (a compound with a very high standard free energy of hydrolysis) (see Fig. 13–3, Table 13–6). Although 2-phosphoglycerate and phosphoenolpyruvate contain nearly the same total amount of energy, the loss of the water molecule from 2-phosphoglycerate causes a re- distribution of energy within the molecule, greatly increasing the standard free energy of hydrolysis of the phosphoryl group. 10 Transfer of the Phosphoryl Group from Phosphoenolpyru- vate to ADP The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K H11001 and either Mg 2H11001 or Mn 2H11001 : In this substrate-level phosphorylation, the product pyruvate first appears in its enol form, then tautomer- izes rapidly and nonenzymatically to its keto form, which predominates at pH 7: The overall reaction has a large, negative standard free- energy change, due in large part to the spontaneous con- version of the enol form of pyruvate to the keto form (see Fig. 13–3). The H9004GH11032H11034 of phosphoenolpyruvate O H11002H11002 C CH 3 O tautomerization O C CH 2 Pyruvate (enol form) Pyruvate (keto form) O O OH CC Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway532 2H11002 O 3 P 2H11002 O 3 P His N NH H11001 COO H11002 HCOH CH 2 OPO 3 2H11002 3-Phosphoglycerate COO H11002 HCOPO 3 2H11002 CH 2 OPO 3 2H11002 2,3-Bisphosphoglycerate (2,3-BPG) COO H11002 HCOPO 3 2H11002 CH 2 OH 2-Phosphoglycerate His N NH H11001 Phosphoglycerate mutase 1 2 His HN NH H11001 FIGURE 14–8 The phosphoglycerate mutase reaction. The enzyme is initially phosphorylated on a His residue. 1 The phosphoenzyme transfers its phosphoryl group to 3-phosphoglycerate, forming 2,3- BPG. 2 The phosphoryl group at C-3 of 2,3-BPG is transferred to the same His residue on the enzyme, producing 2-phosphoglycerate and regenerating the phosphoenzyme. 7.5 kJ/molH11005H9004GH11032H11034 O H11002H11002 C CH 2 H 2 O enolase O CH CH 2 2-Phosphoglycerate Phosphoenolpyruvate O O HO C C OPO 3 2H11002 OPO 3 2H11002 H11005H1100231.4 kJ/molH9004GH11032° P O P O O P H11002 H11002 H11002 H11002 O C C CH 3 H11001 OO O P AdenineRib , K H11001 pyruvate kinase O O C C CH 2 P P AdenineRib O H11002 H11001 ATP Phosphoenolpyruvate Pyruvate ADP O H11002 O Mg 2H11001 O O 8885d_c14_532 2/9/04 7:04 AM Page 532 mac76 mac76:385_reb: hydrolysis is H1100261.9 kJ/mol; about half of this energy is conserved in the formation of the phosphoanhydride bond of ATP (H9004GH11032H11034 H11005 H1100230.5 kJ/mol), and the rest (H1100231.4 kJ/mol) constitutes a large driving force push- ing the reaction toward ATP synthesis. The pyruvate kinase reaction is essentially irreversible under intra- cellular conditions and is an important site of regula- tion, as described in Chapter 15. The Overall Balance Sheet Shows a Net Gain of ATP We can now construct a balance sheet for glycolysis to account for (1) the fate of the carbon skeleton of glu- cose, (2) the input of P i and ADP and the output of ATP, and (3) the pathway of electrons in the oxidation- reduction reactions. The left-hand side of the following equation shows all the inputs of ATP, NAD H11001 , ADP, and P i (consult Fig. 14–2), and the right-hand side shows all the outputs (keep in mind that each molecule of glucose yields two molecules of pyruvate): Glucose H11001 2ATP H11001 2NAD H11001 H11001 4ADP H11001 2P i 88n 2 pyruvate H11001 2ADP H11001 2NADH H11001 2H H11001 H11001 4ATP H11001 2H 2 O Canceling out common terms on both sides of the equa- tion gives the overall equation for glycolysis under aer- obic conditions: Glucose H11001 2NAD H11001 H11001 2ADP H11001 2P i 88n 2 pyruvate H11001 2NADH H11001 2H H11001 H11001 2ATP H11001 2H 2 O The two molecules of NADH formed by glycolysis in the cytosol are, under aerobic conditions, reoxidized to NAD H11001 by transfer of their electrons to the electron- transfer chain, which in eukaryotic cells is located in the mitochondria. The electron-transfer chain passes these electrons to their ultimate destination, O 2 : 2NADH H11001 2H H11001 H11001 O 2 88n 2NAD H11001 H11001 2H 2 O Electron transfer from NADH to O 2 in mitochondria pro- vides the energy for synthesis of ATP by respiration- linked phosphorylation (Chapter 19). In the overall glycolytic process, one molecule of glucose is converted to two molecules of pyruvate (the pathway of carbon). Two molecules of ADP and two of P i are converted to two molecules of ATP (the pathway of phosphoryl groups). Four electrons, as two hydride ions, are transferred from two molecules of glyceralde- hyde 3-phosphate to two of NAD H11001 (the pathway of elec- trons). Glycolysis Is under Tight Regulation During his studies on the fermentation of glucose by yeast, Louis Pasteur discovered that both the rate and the total amount of glucose consumption were many times greater under anaerobic than aerobic conditions. Later studies of muscle showed the same large differ- ence in the rates of anaerobic and aerobic glycolysis. The biochemical basis of this “Pasteur effect” is now clear. The ATP yield from glycolysis under anaerobic conditions (2 ATP per molecule of glucose) is much smaller than that from the complete oxidation of glu- cose to CO 2 under aerobic conditions (30 or 32 ATP per glucose; see Table 19–5). About 15 times as much glu- cose must therefore be consumed anaerobically as aer- obically to yield the same amount of ATP. The flux of glucose through the glycolytic pathway is regulated to maintain nearly constant ATP levels (as well as adequate supplies of glycolytic intermediates that serve biosynthetic roles). The required adjustment in the rate of glycolysis is achieved by a complex inter- play among ATP consumption, NADH regeneration, and allosteric regulation of several glycolytic enzymes—in- cluding hexokinase, PFK-1, and pyruvate kinase—and by second-to-second fluctuations in the concentration of key metabolites that reflect the cellular balance be- tween ATP production and consumption. On a slightly longer time scale, glycolysis is regulated by the hor- mones glucagon, epinephrine, and insulin, and by changes in the expression of the genes for several gly- colytic enzymes. We return to a more detailed discus- sion of the regulation of glycolysis in Chapter 15. Cancerous Tissue Has Deranged Glucose Catabolism Glucose uptake and glycolysis proceed about ten times faster in most solid tumors than in non- cancerous tissues. Tumor cells commonly experience hypoxia (limited oxygen supply), because they initially lack an extensive capillary network to supply the tumor with oxygen. As a result, cancer cells more than 100 to 200 H9262m from the nearest capillaries depend on anaero- bic glycolysis for much of their ATP production. They take up more glucose than normal cells, converting it to pyruvate and then to lactate as they recycle NADH. The high glycolytic rate may also result in part from smaller numbers of mitochondria in tumor cells; less ATP made by respiration-linked phosphorylation in mitochondria means more ATP is needed from glycolysis. In addition, some tumor cells overproduce several glycolytic en- zymes, including an isozyme of hexokinase that associ- ates with the cytosolic face of the mitochondrial inner membrane and is insensitive to feedback inhibition by glucose 6-phosphate. This enzyme may monopolize the ATP produced in mitochondria, using it to convert glu- cose to glucose 6-phosphate and committing the cell to continued glycolysis. The hypoxia-inducible transcrip- tion factor (HIF-1) is a protein that acts at the level of mRNA synthesis to stimulate the synthesis of at least eight of the glycolytic enzymes. This gives the tumor cell the capacity to survive anaerobic conditions until the supply of blood vessels has caught up with tumor growth. 14.1 Glycolysis 533 8885d_c14_521-559 2/6/04 3:43 PM Page 533 mac76 mac76:385_reb: The German biochemist Otto Warburg was the first to show, as early as 1928, that tumors have a higher rate of glucose metabolism than other tissues. With his as- sociates, Warburg purified and crystallized seven of the enzymes of glycolysis. In these studies he developed and used an experimental tool that revolutionized biochem- ical studies of oxidative metabolism: the Warburg manometer, which measured directly the consumption of oxygen by monitoring changes in gas volume, and therefore allowed quantitative measurement of any en- zyme with oxidase activity. Warburg, considered by many the preeminent bio- chemist of the first half of the twentieth century, made seminal contributions to many other areas of biochemistry, including respiration, photo- synthesis, and the enzymol- ogy of intermediary metabo- lism. Trained in carbohydrate chemistry in the laboratory of the great Emil Fischer (who won the Nobel Prize in Chem- istry in 1902), Warburg him- self won the Nobel Prize in Physiology or Medicine in 1931. A number of Warburg’s students and colleagues also were awarded Nobel Prizes: Otto Meyerhof in 1922, Hans Krebs and Fritz Lipmann in 1953, and Hugo Theorell in 1955. Meyerhof’s labora- tory provided training for Lipmann, and for several other Nobel Prize winners: Severo Ochoa (1959), Andre Lwoff (1965), and George Wald (1967). ■ SUMMARY 14.1 Glycolysis ■ Glycolysis is a near-universal pathway by which a glucose molecule is oxidized to two molecules of pyruvate, with energy conserved as ATP and NADH. ■ All ten glycolytic enzymes are in the cytosol, and all ten intermediates are phosphorylated compounds of three or six carbons. ■ In the preparatory phase of glycolysis, ATP is invested to convert glucose to fructose 1,6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate. ■ In the payoff phase, each of the two molecules of glyceraldehyde 3-phosphate derived from glucose undergoes oxidation at C-1; the energy of this oxidation reaction is conserved in the formation of one NADH and two ATP per triose phosphate oxidized. The net equation for the overall process is Glucose H11001 2NAD H11001 H11001 2ADP H11001 2P i 88n 2 pyruvate H11001 2NADH H11001 2H H11001 H11001 2ATP H11001 2H 2 O ■ Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP. Hexokinase, PFK-1, and pyruvate kinase are all subject to allosteric regulation that controls the flow of carbon through the pathway and maintains constant levels of metabolic intermediates. 14.2 Feeder Pathways for Glycolysis Many carbohydrates besides glucose meet their cata- bolic fate in glycolysis, after being transformed into one of the glycolytic intermediates. The most significant are the storage polysaccharides glycogen and starch; the disaccharides maltose, lactose, trehalose, and sucrose; and the monosaccharides fructose, mannose, and galac- tose (Fig. 14–9). Glycogen and Starch Are Degraded by Phosphorolysis Glycogen in animal tissues and in microorganisms (and starch in plants) can be mobilized for use within the same cell by a phosphorolytic reaction catalyzed by glycogen phosphorylase (starch phosphorylase in plants). These enzymes catalyze an attack by P i on the (H92511n4) glycosidic linkage that joins the last two glu- cose residues at a nonreducing end, generating glucose 1-phosphate and a polymer one glucose unit shorter (Fig. 14–10). Phosphorolysis preserves some of the en- ergy of the glycosidic bond in the phosphate ester glu- cose 1-phosphate. Glycogen phosphorylase (or starch phosphorylase) acts repetitively until it approaches an (H92511n6) branch point (see Fig. 7–15), where its action stops. A debranching enzyme removes the branches. The mechanisms and control of glycogen degradation are described in detail in Chapter 15. Glucose 1-phosphate produced by glycogen phos- phorylase is converted to glucose 6-phosphate by phosphoglucomutase, which catalyzes the reversible reaction Glucose 1-phosphate glucose 6-phosphate The glucose 6-phosphate thus formed can enter glycol- ysis or another pathway such as the pentose phosphate pathway, described in Section 14.5. Phosphoglucomu- tase employs essentially the same mechanism as phos- phoglycerate mutase (p. 531). The general name mu- tase is given to enzymes that catalyze the transfer of a functional group from one position to another in the same molecule. Mutases are a subclass of isomerases, enzymes that interconvert stereoisomers or structural or positional isomers (see Table 6–3). z y Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway534 Otto Warburg, 1883–1970 8885d_c14_521-559 2/6/04 3:43 PM Page 534 mac76 mac76:385_reb: Dietary Polysaccharides and Disaccharides Undergo Hydrolysis to Monosaccharides For most humans, starch is the major source of carbo- hydrates in the diet. Digestion begins in the mouth, where salivary H9251-amylase (Fig. 14–9) hydrolyzes the in- ternal glycosidic linkages of starch, producing short poly- saccharide fragments or oligosaccharides. (Note that in this hydrolysis reaction, water, not P i , is the attacking species.) In the stomach, salivary H9251-amylase is inacti- vated by the low pH, but a second form of H9251-amylase, secreted by the pancreas into the small intestine, con- tinues the breakdown process. Pancreatic H9251-amylase yields mainly maltose and maltotriose (the di- and trisac- charides of H9251(1n4) glucose) and oligosaccharides called limit dextrins, fragments of amylopectin containing H9251(1n6) branch points. Maltose and dextrins are de- graded by enzymes of the intestinal brush border (the fingerlike microvilli of intestinal epithelial cells, which greatly increase the area of the intestinal surface). Di- etary glycogen has essentially the same structure as starch, and its digestion proceeds by the same pathway. Disaccharides must be hydrolyzed to monosaccha- rides before entering cells. Intestinal disaccharides and dextrins are hydrolyzed by enzymes attached to the outer surface of the intestinal epithelial cells: 14.2 Feeder Pathways for Glycolysis 535 OH HH H OH CH 2 OH Glyceraldehyde 3-phosphate sucrase fructose 1- phosphate aldolase UDP-galactose P H 2 O i CH 2 OH O H H H OH HO D-Fructose OH O HO H H H H OH H CH 2 OH D-Glucose OH OH OH HOCH 2 D-Mannose Glycogen; starch Glucose 1-phosphate Lactose Mannose 6-phosphate Glucose 6-phosphate Sucrose Trehalose phosphogluco- mutase lactase H9251-amylase trehalase UDP-glucose O HO H H H H OH H CH 2 OH OH OH D-Galactose H O OH H ATP hexokinase ATP ATP phosphomannose isomerase Fructose 1,6- bisphosphate triose phosphate isomerase Fructose 1-phosphate phosphate ATP fructokinase Glyceraldehyde H11001 Dihydroxyacetone triose kinase hexokinase ATP Fructose 6-phosphate hexokinase phosphorylase FIGURE 14–9 Entry of glycogen, starch, disaccharides, and hexoses into the preparatory stage of glycolysis. Dextrin H11001 nH 2 O 8888888n n D-glucose dextrinase Maltose H11001 H 2 O 8888888n 2 D-glucose maltase Lactose H11001 H 2 O 8888888n D-galactose H11001 D-glucose lactase Sucrose H11001 H 2 O 8888888n D-fructose H11001 D-glucose sucrase Trehalose H11001 H 2 O 8888888n 2 D-glucose trehalase The monosaccharides so formed are actively trans- ported into the epithelial cells (see Fig. 11–44), then passed into the blood to be carried to various tissues, where they are phosphorylated and funneled into the glycolytic sequence. Lactose intolerance, common among adults of most human populations except those originating 8885d_c14_521-559 2/6/04 3:43 PM Page 535 mac76 mac76:385_reb: in Northern Europe and some parts of Africa, is due to the disappearance after childhood of most or all of the lactase activity of the intestinal cells. Lactose cannot be completely digested and absorbed in the small intestine and passes into the large intestine, where bacteria con- vert it to toxic products that cause abdominal cramps and diarrhea. The problem is further complicated be- cause undigested lactose and its metabolites increase the osmolarity of the intestinal contents, favoring the retention of water in the intestine. In most parts of the world where lactose intolerance is prevalent, milk is not used as a food by adults, although milk products predi- gested with lactase are commercially available in some countries. In certain human disorders, several or all of the intestinal disaccharidases are missing. In these cases, the digestive disturbances triggered by dietary disaccharides can sometimes be minimized by a con- trolled diet. ■ Other Monosaccharides Enter the Glycolytic Pathway at Several Points In most organisms, hexoses other than glucose can un- dergo glycolysis after conversion to a phosphorylated derivative. D-Fructose, present in free form in many fruits and formed by hydrolysis of sucrose in the small intestine of vertebrates, is phosphorylated by hexokinase: Mg 2H11001 Fructose H11001 ATP 88n fructose 6-phosphate H11001 ADP This is a major pathway of fructose entry into glycoly- sis in the muscles and kidney. In the liver, however, fruc- tose enters by a different pathway. The liver enzyme fructokinase catalyzes the phosphorylation of fructose at C-1 rather than C-6: Mg 2H11001 Fructose H11001 ATP 88n fructose 1-phosphate H11001 ADP The fructose 1-phosphate is then cleaved to glycer- aldehyde and dihydroxyacetone phosphate by fructose 1-phosphate aldolase: Dihydroxyacetone phosphate is converted to glycer- aldehyde 3-phosphate by the glycolytic enzyme triose phosphate isomerase. Glyceraldehyde is phosphorylated by ATP and triose kinase to glyceraldehyde 3-phos- phate: Mg 2H11001 Glyceraldehyde H11001 ATP On glyceraldehyde 3-phosphate H11001 ADP Thus both products of fructose 1-phosphate hydrolysis enter the glycolytic pathway as glyceraldehyde 3- phosphate. D-Galactose, a product of hydrolysis of the dis- accharide lactose (milk sugar), passes in the blood from the intestine to the liver, where it is first phosphorylated at C-1, at the expense of ATP, by the enzyme galactokinase: Mg 2H11001 Galactose H11001 ATP 88n galactose 1-phosphate H11001 ADP The galactose 1-phosphate is then converted to its epimer at C-4, glucose 1-phosphate, by a set of reac- tions in which uridine diphosphate (UDP) functions as a coenzyme-like carrier of hexose groups (Fig. 14–11). The epimerization involves first the oxidation of the C-4 OOH group to a ketone, then reduction of the ketone to an OOH, with inversion of the configuration at C-4. NAD is the cofactor for both the oxidation and the reduction. O H11001 A A P A A A HCOH A HCOH CH 2 OH A HCOH Glyceraldehyde fructose 1-phosphate aldolase H PO CH 2 OH Fructose 1-phosphate Dihydroxyacetone phosphate C C CH 2 OH O HOCH C A P A A 1 2 3 4 5 6 CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway536 HO OH OH O H11002 O H11002 OH O O O H H H H H P Glycogen (starch) n glucose units glycogen (starch) phosphorylase O H11002 O H11002 OP Nonreducing end CH 2 OH OH OH O O H H H H H H CH 2 OH HO OH OH O O H H H H H Glycogen (starch) (nH110021) glucose units HO CH 2 OH OH OH O O H H H H H H CH 2 OH H11001 Glucose 1-phosphate FIGURE 14–10 Glycogen breakdown by glycogen phosphorylase. The enzyme catalyzes attack by inorganic phosphate (pink) on the ter- minal glucosyl residue (blue) at the nonreducing end of a glycogen molecule, releasing glucose 1-phosphate and generating a glycogen molecule shortened by one glucose residue. The reaction is a phos- phorolysis (not hydrolysis). 8885d_c14_521-559 2/6/04 3:43 PM Page 536 mac76 mac76:385_reb: Defects in any of the three enzymes in this pathway cause galactosemia in humans. In galactokinase- deficiency galactosemia, high galactose concentrations are found in blood and urine. Infants develop cataracts, caused by deposition of the galactose metabolite galac- titol in the lens. The symptoms in this disorder are relatively mild, and strict limitation of galactose in the diet greatly dimin- ishes their severity. Transferase-deficiency galactosemia is more seri- ous; it is characterized by poor growth in children, speech abnormality, mental deficiency, and liver dam- age that may be fatal, even when galactose is withheld from the diet. Epimerase-deficiency galactosemia leads to similar symptoms, but is less severe when dietary galactose is carefully controlled. ■ D-Mannose, released in the digestion of various poly- saccharides and glycoproteins of foods, can be phos- phorylated at C-6 by hexokinase: Mg 2H11001 Mannose H11001 ATP 88n mannose 6-phosphate H11001 ADP Mannose 6-phosphate is isomerized by phosphoman- nose isomerase to yield fructose 6-phosphate, an in- termediate of glycolysis. SUMMARY 14.2 Feeder Pathways for Glycolysis ■ Glycogen and starch, polymeric storage forms of glucose, enter glycolysis in a two-step process. Phosphorolytic cleavage of a glucose residue from an end of the polymer, forming glucose 1-phosphate, is catalyzed by glycogen phosphorylase or starch phosphorylase. Phosphoglucomutase then converts the glucose 1-phosphate to glucose 6-phosphate, which can enter glycolysis. ■ Ingested polysaccharides and disaccharides are converted to monosaccharides by intestinal hydrolytic enzymes, and the monosaccharides then enter intestinal cells and are transported to the liver or other tissues. ■ A variety of D-hexoses, including fructose, galactose, and mannose, can be funneled into glycolysis. Each is phosphorylated and converted to either glucose 6-phosphate or fructose 6-phosphate. ■ Conversion of galactose 1-phosphate to glucose 1-phosphate involves two nucleotide derivatives: UDP-galactose and UDP-glucose. Genetic de- fects in any of the three enzymes that catalyze conversion of galactose to glucose 1-phosphate result in galactosemias of varying severity. CH 2 OH CH 2 OH OHH C D-Galactitol OHH C HO HC HO HC 537 UDP H H H OH O CH 2 OH O HO H OH H H H OH H O O O CH 2 OH P O OU O H OH Glucose 1-phosphate UDP-glucose: galactose 1- phosphate uridylyltransferase Mg 2H11001 UDP-glucose galactokinase ADP ATP Galactose UDP- glucose HO H H HO H H H OH CH 2 OH O H OH Galactose 1-phosphate UDP-galactose 4 4 UDP H H H OH O CH 2 OH O O H OH UDP NAD H11001 NADH H11001 H H11001 UDP-glucose 4-epimerase NAD H11001 NADH H11001 H H11001 UDP-glucose 4-epimerase FIGURE 14–11 Conversion of galactose to glucose 1-phosphate. The conversion proceeds through a sugar-nucleotide derivative, UDP- galactose, which is formed when galactose 1-phosphate displaces glu- cose 1-phosphate from UDP-glucose. UDP-galactose is then converted by UDP-glucose 4-epimerase to UDP-glucose, in a reaction that in- volves oxidation of C-4 (pink) by NAD H11001 , then reduction of C-4 by NADH; the result is inversion of the configuration at C-4. The UDP- glucose is recycled through another round of the same reaction. The net effect of this cycle is the conversion of galactose 1-phosphate to glucose 1-phosphate; there is no net production or consumption of UDP-galactose or UDP-glucose. 14.2 Feeder Pathways for Glycolysis 8885d_c14_521-559 2/6/04 3:43 PM Page 537 mac76 mac76:385_reb: 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation Pyruvate occupies an important junction in carbohy- drate catabolism (Fig. 14–3). Under aerobic conditions pyruvate is oxidized to acetate, which enters the citric acid cycle and is oxidized to CO 2 and H 2 O, and NADH formed by the dehydrogenation of glyceraldehyde 3- phosphate is ultimately reoxidized to NAD H11001 by passage of its electrons to O 2 in mitochondrial respiration. How- ever, under hypoxic conditions, as in very active skele- tal muscle, in submerged plant tissues, or in lactic acid bacteria, NADH generated by glycolysis cannot be re- oxidized by O 2 . Failure to regenerate NAD H11001 would leave the cell with no electron acceptor for the oxidation of glyceraldehyde 3-phosphate, and the energy-yielding reactions of glycolysis would stop. NAD H11001 must there- fore be regenerated in some other way. The earliest cells lived in an atmosphere almost devoid of oxygen and had to develop strategies for de- riving energy from fuel molecules under anaerobic conditions. Most modern organisms have retained the ability to constantly regenerate NAD H11001 during anaero- bic glycolysis by transferring electrons from NADH to form a reduced end product such as lactate or ethanol. Pyruvate Is the Terminal Electron Acceptor in Lactic Acid Fermentation When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD H11001 is regenerated from NADH by the reduction of pyruvate to lactate. As mentioned earlier, some tissues and cell types (such as erythrocytes, which have no mitochondria and thus can- not oxidize pyruvate to CO 2 ) produce lactate from glu- cose even under aerobic conditions. The reduction of pyruvate is catalyzed by lactate dehydrogenase, which forms the L isomer of lactate at pH 7: The overall equilibrium of this reaction strongly favors lactate formation, as shown by the large negative standard free-energy change. In glycolysis, dehydrogenation of the two molecules of glyceraldehyde 3-phosphate derived from each mol- ecule of glucose converts two molecules of NAD H11001 to two of NADH. Because the reduction of two molecules of pyruvate to two of lactate regenerates two molecules of NAD H11001 , there is no net change in NAD H11001 or NADH: O H11002 H11002 H11001 C CH 3 HO NAD O C CH 3 Pyruvate O O O C C lactate dehydrogenase H11001 NADH H11001 H H 25.1 kJ/mol L-Lactate H11005 H11002H9004GH11032H11034 The lactate formed by active skeletal muscles (or by ery- throcytes) can be recycled; it is carried in the blood to the liver, where it is converted to glucose during the re- covery from strenuous muscular activity. When lactate is produced in large quantities during vigorous muscle contraction (during a sprint, for example), the acidifi- cation that results from ionization of lactic acid in mus- cle and blood limits the period of vigorous activity. The best-conditioned athletes can sprint at top speed for no more than a minute (Box 14–1). Although conversion of glucose to lactate includes two oxidation-reduction steps, there is no net change in the oxidation state of carbon; in glucose (C 6 H 12 O 6 ) and lactic acid (C 3 H 6 O 3 ), the H:C ratio is the same. Never- theless, some of the energy of the glucose molecule has been extracted by its conversion to lactate—enough to give a net yield of two molecules of ATP for every glu- cose molecule consumed. Fermentation is the general term for such processes, which extract energy (as ATP) but do not consume oxygen or change the concentra- tions of NAD H11001 or NADH. Fermentations are carried out by a wide range of organisms, many of which occupy anaerobic niches, and they yield a variety of end prod- ucts, some of which find commercial uses. Ethanol Is the Reduced Product in Ethanol Fermentation Yeast and other microorganisms ferment glucose to ethanol and CO 2 , rather than to lactate. Glucose is con- verted to pyruvate by glycolysis, and the pyruvate is converted to ethanol and CO 2 in a two-step process: In the first step, pyruvate is decarboxylated in an irre- versible reaction catalyzed by pyruvate decarboxy- lase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate de- carboxylase requires Mg 2H11001 and has a tightly bound coenzyme, thiamine pyrophosphate, discussed below. In the second step, acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase, with 2 Pyruvate 2 Lactate Glucose 2NADH 2NAD H11001 Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway538 O OH NAD O C CH 3 Pyruvate H O C alcohol dehydrogenase NADH H11001 HCO 2 TPP, Mg 2H11001 CH 3 Acetaldehyde pyruvate decarboxylase O H11002 H11001 H11001 C CH 2 CH 3 Ethanol 8885d_c14_538 2/9/04 7:04 AM Page 538 mac76 mac76:385_reb: 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation 539 BOX 14–1 THE WORLD OF BIOCHEMISTRY Athletes, Alligators, and Coelacanths: Glycolysis at Limiting Concentrations of Oxygen Most vertebrates are essentially aerobic organisms; they convert glucose to pyruvate by glycolysis, then use molecular oxygen to oxidize the pyruvate com- pletely to CO 2 and H 2 O. Anaerobic catabolism of glu- cose to lactate occurs during short bursts of extreme muscular activity, for example in a 100 m sprint, dur- ing which oxygen cannot be carried to the muscles fast enough to oxidize pyruvate. Instead, the muscles use their stored glucose (glycogen) as fuel to gener- ate ATP by fermentation, with lactate as the end prod- uct. In a sprint, lactate in the blood builds up to high concentrations. It is slowly converted back to glucose by gluconeogenesis in the liver in the subsequent rest or recovery period, during which oxygen is consumed at a gradually diminishing rate until the breathing rate returns to normal. The excess oxygen consumed in the recovery period represents a repayment of the oxygen debt. This is the amount of oxygen required to supply ATP for gluconeogenesis during recovery respiration, in order to regenerate the glycogen “bor- rowed” from liver and muscle to carry out intense mus- cular activity in the sprint. The cycle of reactions that includes glucose conversion to lactate in muscle and lactate conversion to glucose in liver is called the Cori cycle, for Carl and Gerty Cori, whose studies in the 1930s and 1940s clarified the pathway and its role (see Box 15–1). The circulatory systems of most small vertebrates can carry oxygen to their muscles fast enough to avoid having to use muscle glycogen anaerobically. For ex- ample, migrating birds often fly great distances at high speeds without rest and without incurring an oxygen debt. Many running animals of moderate size also main- tain an essentially aerobic metabolism in their skele- tal muscle. However, the circulatory systems of larger animals, including humans, cannot completely sustain aerobic metabolism in skeletal muscles over long pe- riods of intense muscular activity. These animals gen- erally are slow-moving under normal circumstances and engage in intense muscular activity only in the gravest emergencies, because such bursts of activity require long recovery periods to repay the oxygen debt. Alligators and crocodiles, for example, are nor- mally sluggish animals. Yet when provoked they are capable of lightning-fast charges and dangerous lash- ings of their powerful tails. Such intense bursts of ac- tivity are short and must be followed by long periods of recovery. The fast emergency movements require lactic acid fermentation to generate ATP in skeletal muscles. The stores of muscle glycogen are rapidly ex- pended in intense muscular activity, and lactate reaches very high concentrations in muscles and ex- tracellular fluid. Whereas a trained athlete can recover from a 100 m sprint in 30 min or less, an alligator may require many hours of rest and extra oxygen con- sumption to clear the excess lactate from its blood and regenerate muscle glycogen after a burst of activity. Other large animals, such as the elephant and rhi- noceros, have similar metabolic characteristics, as do diving mammals such as whales and seals. Dinosaurs and other huge, now-extinct animals probably had to depend on lactic acid fermentation to supply energy for muscular activity, followed by very long recovery periods during which they were vulnerable to attack by smaller predators better able to use oxygen and thus better adapted to continuous, sustained muscu- lar activity. Deep-sea explorations have revealed many species of marine life at great ocean depths, where the oxygen concentration is near zero. For example, the primitive coelacanth, a large fish recovered from depths of 4,000 m or more off the coast of South Africa, has an essentially anaerobic metabolism in vir- tually all its tissues. It converts carbohydrates to lac- tate and other products, most of which must be ex- creted. Some marine vertebrates ferment glucose to ethanol and CO 2 in order to generate ATP. 8885d_c14_521-559 2/6/04 3:43 PM Page 539 mac76 mac76:385_reb: the reducing power furnished by NADH derived from the dehydrogenation of glyceraldehyde 3-phosphate. This reaction is a well-studied case of hydride transfer from NADH (Fig. 14–12). Ethanol and CO 2 are thus the end products of ethanol fermentation, and the overall equation is Glucose H11001 2ADP H11001 2P i 88n 2 ethanol H11001 2CO 2 H11001 2ATP H11001 2H 2 O As in lactic acid fermentation, there is no net change in the ratio of hydrogen to carbon atoms when glucose (H:C ratio H11005 12/6 H11005 2) is fermented to two ethanol and Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway540 N HH : C O NH 2 NADH NAD + Ethanol Acetaldehyde CH 3 H + R H N + R C O NH 2 + CZn 2+ O C H H OHCH 3 H Alcohol dehydrogenase MECHANISM FIGURE 14–12 The alcohol dehydrogenase reaction. AZn 2H11001 at the active site polarizes the carbonyl oxygen of acetaldehyde, allowing transfer of a hydride ion (red) from the reduced cofactor NADH. The reduced intermediate acquires a proton from the medium (blue) to form ethanol. Alcohol Dehydrogenase Mechanism TABLE 14–1 Some TPP-Dependent Reactions Enzyme Pathway(s) Bond cleaved Bond formed Pyruvate decarboxylase Ethanol fermentation Pyruvate dehydrogenase Synthesis of acetyl-CoA H9251-Ketoglutarate dehydrogenase Citric acid cycle Transketolase Carbon-assimilation reactions Pentose phosphate pathway R 3 R 5 CC H OOH R 3 R 4 C C H O OH R 2 S-CoA C O R 2 C C O O O H11002 R 1 C H O R 1 CC O O O H11002 ? ? ? ? ? ? ? ? ? ? two CO 2 (combined H:C ratio H11005 12/6 H11005 2). In all fer- mentations, the H:C ratio of the reactants and products remains the same. Pyruvate decarboxylase is present in brewer’s and baker’s yeast and in all other organisms that ferment glucose to ethanol, including some plants. The CO 2 pro- duced by pyruvate decarboxylation in brewer’s yeast is responsible for the characteristic carbonation of cham- pagne. The ancient art of brewing beer involves a num- ber of enzymatic processes in addition to the reactions of ethanol fermentation (Box 14–2). In baking, CO 2 re- leased by pyruvate decarboxylase when yeast is mixed with a fermentable sugar causes dough to rise. The en- zyme is absent in vertebrate tissues and in other or- ganisms that carry out lactic acid fermentation. Alcohol dehydrogenase is present in many organ- isms that metabolize ethanol, including humans. In hu- man liver it catalyzes the oxidation of ethanol, either in- gested or produced by intestinal microorganisms, with the concomitant reduction of NAD H11001 to NADH. Thiamine Pyrophosphate Carries “Active Acetaldehyde” Groups The pyruvate decarboxylase reaction provides our first encounter with thiamine pyrophosphate (TPP) (Fig. 14–13), a coenzyme derived from vitamin B 1 . Lack of vi- tamin B 1 in the human diet leads to the condition known as beriberi, characterized by an accumulation of body fluids (swelling), pain, paralysis, and ultimately death. Thiamine pyrophosphate plays an important role in the cleavage of bonds adjacent to a carbonyl group, such as the decarboxylation of H9251-keto acids, and in chemical rearrangements in which an activated acetaldehyde group is transferred from one carbon atom to another (Table 14–1). The functional part of TPP, the thiazolium ring, has a relatively acidic proton at C-2. Loss of this 8885d_c14_521-559 2/6/04 3:43 PM Page 540 mac76 mac76:385_reb: OP NH 2 CH 2 O H11002 H11002H11002 H11002 H11002H11002 (a) (b) thiazolium ring active acetaldehyde CH 3 N CH 2 N CH 2 O O O P OH O C C CH 3 NH 2 CH 2 N N H CH 3 54 3 2 1 H O Thiamine pyrophosphate (TPP) Hydroxyethyl thiamine pyrophosphate N H11001 H11001 CH 3 S OPOCH 2 CH 2 O O O P O C O N CH 3 S R Acetaldehyde R resonance stabilization CH 3 C H O CH 3 OHCH 3 N . . (c) OHC H CH 3 C CO 2 O OH C 1 2 4 3 Hydroxyethyl TPP C CH 3 OHC C CH 3 S R R C N CH 3 S R R C N CH 3 S R R C N CH 3 S H O H H11001 H11001 H11001 H11001 H11001 H11032H11032 H11032H11032 H11002 H11002 C Pyruvate TPP carbanionTPP 5 R R N CH 3 S R R C H N CH 3 S H11002 H11002 C C O CH 3 O O H11001 H11001 H11032H11032 H H11001 H H11001 proton produces a carbanion that is the active species in TPP-dependent reactions (Fig. 14–13). The carban- ion readily adds to carbonyl groups, and the thiazolium ring is thereby positioned to act as an “electron sink” that greatly facilitates reactions such as the decarboxy- lation catalyzed by pyruvate decarboxylase. Fermentations Yield a Variety of Common Foods and Industrial Chemicals Our progenitors learned millennia ago to use fermenta- tion in the production and preservation of foods. Cer- tain microorganisms present in raw food products fer- ment the carbohydrates and yield metabolic products that give the foods their characteristic forms, textures, and tastes. Yogurt, already known in Biblical times, is produced when the bacterium Lactobacillus bulgari- cus ferments the carbohydrate in milk, producing lac- tic acid; the resulting drop in pH causes the milk pro- teins to precipitate, producing the thick texture and sour taste of unsweetened yogurt. Another bacterium, Propionibacterium freudenreichii, ferments milk to produce propionic acid and CO 2 ; the propionic acid pre- cipitates milk proteins, and bubbles of CO 2 cause the holes characteristic of Swiss cheese. Many other food products are the result of fermentations: pickles, sauer- kraut, sausage, soy sauce, and a variety of national fa- vorites, such as kimchi (Korea), tempoyak (Indonesia), kefir (Russia), dahi (India), and pozol (Mexico). The drop in pH associated with fermentation also helps to preserve foods, because most of the microorganisms that cause food spoilage cannot grow at low pH. In agriculture, plant byproducts such as corn stalks are preserved for use as animal feed by packing them into a large container (a silo) with limited access to air; microbial fermentation produces acids that lower the pH. The silage that results from this fermentation 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation 541 MECHANISM FIGURE 14–13 Thiamine pyrophosphate (TPP) and its role in pyruvate decarboxylation. (a) TPP is the coenzyme form of vi- tamin B 1 (thiamine). The reactive carbon atom in the thiazolium ring of TPP is shown in red. In the reaction catalyzed by pyruvate decar- boxylase, two of the three carbons of pyruvate are carried transiently on TPP in the form of a hydroxyethyl, or “active acetaldehyde,” group (b), which is subsequently released as acetaldehyde. (c) After cleavage of a carbon–carbon bond, one product often has a free electron pair, or carbanion, which because of its strong tendency to form a new bond is generally unstable. The thiazolium ring of TPP stabilizes carbanion intermediates by providing an electrophilic (electron-deficient) struc- ture into which the carbanion electrons can be delocalized by reso- nance. Structures with this property, often called “electron sinks,” play a role in many biochemical reactions. This principle is illustrated here for the reaction catalyzed by pyruvate decarboxylase. 1 The TPP car- banion acts as a nucleophile, attacking the carbonyl group of pyruvate. 2 Decarboxylation produces a carbanion that is stabilized by the thiazolium ring. 3 Protonation to form hydroxyethyl TPP is followed by 4 release of acetaldehyde. 5 A proton dissociates to regenerate the carbanion. Thiamine Pyrophosphate Mechanism 8885d_c14_521-559 2/6/04 3:43 PM Page 541 mac76 mac76:385_reb: process can be kept as animal feed for long periods without spoilage. In 1910 Chaim Weizmann (later to become the first president of Israel) discovered that the bacterium Clostridium acetobutyricum ferments starch to bu- tanol and acetone. This discovery opened the field of industrial fermentations, in which some readily avail- able material rich in carbohydrate (corn starch or mo- lasses, for example) is supplied to a pure culture of a specific microorganism, which ferments it into a prod- uct of greater value. The methanol used to make “gaso- hol” is produced by microbial fermentation, as are formic, acetic, propionic, butyric, and succinic acids, and glycerol, ethanol, isopropanol, butanol, and bu- tanediol. These fermentations are generally carried out in huge closed vats in which temperature and access to air are adjusted to favor the multiplication of the de- sired microorganism and to exclude contaminating organisms (Fig. 14–14). The beauty of industrial fer- mentations is that complicated, multistep chemical transformations are carried out in high yields and with few side products by chemical factories that reproduce themselves—microbial cells. For some industrial fer- mentations, technology has been developed to immobi- lize the cells in an inert support, to pass the starting ma- terial continuously through the bed of immobilized cells, and to collect the desired product in the effluent—an engineer’s dream! Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway542 FIGURE 14–14 Industrial-scale fermentation. Microorganisms are cultured in a sterilizable vessel containing thousands of liters of growth medium—an inexpensive source of both carbon and energy—under carefully controlled conditions, including low oxygen concentration and constant temperature. After centrifugal separation of the cells from the growth medium, the valuable products of the fermentation are re- covered from the cells or from the supernatant fluid. BOX 14–2 THE WORLD OF BIOCHEMISTRY Brewing Beer Brewers prepare beer by ethanol fermentation of the carbohydrates in cereal grains (seeds) such as barley, carried out by yeast glycolytic enzymes. The carbo- hydrates, largely polysaccharides, must first be de- graded to disaccharides and monosaccharides. In a process called malting, the barley seeds are allowed to germinate until they form the hydrolytic enzymes required to break down their polysaccharides, at which point germination is stopped by controlled heat- ing. The product is malt, which contains enzymes that catalyze the hydrolysis of the H9252 linkages of cellulose and other cell wall polysaccharides of the barley husks, and enzymes such as H9251-amylase and maltase. The brewer next prepares the wort, the nutrient medium required for fermentation by yeast cells. The malt is mixed with water and then mashed or crushed. This allows the enzymes formed in the malting process to act on the cereal polysaccharides to form maltose, glucose, and other simple sugars, which are soluble in the aqueous medium. The remaining cell matter is then separated, and the liquid wort is boiled with hops to give flavor. The wort is cooled and then aerated. Now the yeast cells are added. In the aerobic wort the yeast grows and reproduces very rapidly, using en- ergy obtained from available sugars. No ethanol forms during this stage, because the yeast, amply supplied with oxygen, oxidizes the pyruvate formed by glycoly- sis to CO 2 and H 2 O via the citric acid cycle. When all the dissolved oxygen in the vat of wort has been con- sumed, the yeast cells switch to anaerobic metabolism, and from this point they ferment the sugars into ethanol and CO 2 . The fermentation process is controlled in part by the concentration of the ethanol formed, by the pH, and by the amount of remaining sugar. After fermen- tation has been stopped, the cells are removed and the “raw” beer is ready for final processing. In the final steps of brewing, the amount of foam or head on the beer, which results from dissolved pro- teins, is adjusted. Normally this is controlled by pro- teolytic enzymes that arise in the malting process. If these enzymes act on the proteins too long, the beer will have very little head and will be flat; if they do not act long enough, the beer will not be clear when it is cold. Sometimes proteolytic enzymes from other sources are added to control the head. 8885d_c14_521-559 2/6/04 3:43 PM Page 542 mac76 mac76:385_reb: SUMMARY 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation ■ The NADH formed in glycolysis must be recycled to regenerate NAD H11001 , which is required as an electron acceptor in the first step of the payoff phase. Under aerobic conditions, electrons pass from NADH to O 2 in mitochondrial respiration. ■ Under anaerobic or hypoxic conditions, many organisms regenerate NAD H11001 by transferring electrons from NADH to pyruvate, forming lactate. Other organisms, such as yeast, regenerate NAD H11001 by reducing pyruvate to ethanol and CO 2 . In these anaerobic processes (fermentations), there is no net oxidation or reduction of the carbons of glucose. ■ A variety of microorganisms can ferment sugar in fresh foods, resulting in changes in pH, taste, and texture, and preserving food from spoilage. Fermentations are used in industry to produce a wide variety of commercially valuable organic compounds from inexpensive starting materials. 14.4 Gluconeogenesis The central role of glucose in metabolism arose early in evolution, and this sugar remains the nearly universal fuel and building block in modern organisms, from mi- crobes to humans. In mammals, some tissues depend almost completely on glucose for their metabolic energy. For the human brain and nervous system, as well as the erythrocytes, testes, renal medulla, and embryonic tis- sues, glucose from the blood is the sole or major fuel source. The brain alone requires about 120 g of glucose each day—more than half of all the glucose stored as glycogen in muscle and liver. However, the supply of glu- cose from these stores is not always sufficient; between meals and during longer fasts, or after vigorous exer- cise, glycogen is depleted. For these times, organisms need a method for synthesizing glucose from noncar- bohydrate precursors. This is accomplished by a path- way called gluconeogenesis (“formation of new sugar”), which converts pyruvate and related three- and four-carbon compounds to glucose. Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms. The reactions are essentially the same in all tissues and all species. The important pre- cursors of glucose in animals are three-carbon com- pounds such as lactate, pyruvate, and glycerol, as well as certain amino acids (Fig. 14–15). In mammals, glu- coneogenesis takes place mainly in the liver, and to a lesser extent in renal cortex. The glucose produced passes into the blood to supply other tissues. After vig- orous exercise, lactate produced by anaerobic glycoly- sis in skeletal muscle returns to the liver and is con- verted to glucose, which moves back to muscle and is converted to glycogen—a circuit called the Cori cycle (Box 14–1; see also Fig. 23–18). In plant seedlings, stored fats and proteins are converted, via paths that include gluconeogenesis, to the disaccharide sucrose for transport throughout the developing plant. Glucose and its derivatives are precursors for the synthesis of plant cell walls, nucleotides and coenzymes, and a variety of other essential metabolites. In many microorganisms, gluconeogenesis starts from simple organic compounds of two or three carbons, such as acetate, lactate, and propionate, in their growth medium. Although the reactions of gluconeogenesis are the same in all organisms, the metabolic context and the regulation of the pathway differ from one species to an- other and from tissue to tissue. In this section we focus on gluconeogenesis as it occurs in the mammalian liver. In Chapter 20 we show how photosynthetic organisms use this pathway to convert the primary products of photosynthesis into glucose, to be stored as sucrose or starch. 14.4 Gluconeogenesis 543 Glycoproteins Blood glucose Glycogen Glucogenic amino acids Citric acid cycle Glucose 6-phosphate Other monosaccharides Sucrose Disaccharides Pyruvate Lactate Phosphoenol- pyruvate 3-Phospho- glycerate CO 2 fixation Triacyl- glycerols Glycerol Animals Plants Starch Energy FIGURE 14–15 Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is com- mon to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. Plants and photosynthetic bac- teria are uniquely able to convert CO 2 to carbohydrates. 8885d_c14_521-559 2/6/04 3:43 PM Page 543 mac76 mac76:385_reb: Gluconeogenesis and glycolysis are not identical pathways running in opposite directions, although they do share several steps (Fig. 14–16); seven of the ten en- zymatic reactions of gluconeogenesis are the reverse of glycolytic reactions. However, three reactions of glycol- ysis are essentially irreversible in vivo and cannot be used in gluconeogenesis: the conversion of glucose to glucose 6-phosphate by hexokinase, the phosphoryla- tion of fructose 6-phosphate to fructose 1,6-bisphos- phate by phosphofructokinase-1, and the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase (Fig. 14–16). In cells, these three reactions are charac- terized by a large negative free-energy change, H9004G, whereas other glycolytic reactions have a H9004G near 0 (Table 14–2). In gluconeogenesis, the three irreversible steps are bypassed by a separate set of enzymes, cat- alyzing reactions that are sufficiently exergonic to be ef- fectively irreversible in the direction of glucose synthe- sis. Thus, both glycolysis and gluconeogenesis are irreversible processes in cells. In animals, both pathways occur largely in the cytosol, necessitating their recipro- cal and coordinated regulation. Separate regulation of the two pathways is brought about through controls ex- erted on the enzymatic steps unique to each. We begin by considering the three bypass reactions of gluconeogenesis. (Keep in mind that “bypass” refers throughout to the bypass of irreversible glycolytic re- actions.) Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate (PEP). This reaction cannot occur by reversal of the pyruvate kinase reaction of glycolysis (p. 532), which has a large, negative standard free-energy change and is irreversible under the conditions prevailing in intact cells (Table 14–2, step 10). Instead, the phosphoryla- tion of pyruvate is achieved by a roundabout sequence of reactions that in eukaryotes requires enzymes in both the cytosol and mitochondria. As we shall see, the path- way shown in Figure 14–16 and described in detail here is one of two routes from pyruvate to PEP; it is the pre- dominant path when pyruvate or alanine is the gluco- genic precursor. A second pathway, described later, pre- dominates when lactate is the glucogenic precursor. Pyruvate is first transported from the cytosol into mitochondria or is generated from alanine within mito- chondria by transamination, in which the H9251-amino group is removed from alanine (leaving pyruvate) and added to an H9251-keto carboxylic acid (transamination reactions are discussed in detail in Chapter 18). Then pyruvate carboxylase, a mitochondrial enzyme that requires the coenzyme biotin, converts the pyruvate to oxaloacetate (Fig. 14–17): Pyruvate H11001 HCO 3 H11002 H11001 ATP 88n oxaloacetate H11001 ADP H11001 P i (14–4) Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway544 Glycolysis hexokinase ATP ADP Glucose Glucose 6-phosphate ATP ADP H 2 O P i H 2 O P i glucose 6-phosphatase phospho- fructokinase-1 Fructose 6-phosphate Fructose 1,6-bisphosphate fructose 1,6-bisphosphatase Gluconeogenesis (2) P i (2) P i (2) NADH H11001 (2) H H11001 (2) NADH H11001 H H11001 (2) 1,3-Bisphosphoglycerate (2) ADP (2) ATP (2) ADP (2) ATP (2) ATP (2) ADP (2) GDP (2) GTP (2) 3-Phosphoglycerate (2) 2-Phosphoglycerate (2) Phosphoenolpyruvate (2) Pyruvate (2) Oxaloacetate pyruvate carboxylase PEP carboxykinase pyruvate kinase (2) ADP (2) ATP Dihydroxyacetone phosphate Dihydroxyacetone phosphate (2) Glyceraldehyde 3-phosphate (2) NAD H11001 (2) NAD H11001 FIGURE 14–16 Opposing pathways of glycolysis and gluconeogene- sis in rat liver. The reactions of glycolysis are shown on the left side in blue; the opposing pathway of gluconeogenesis is shown on the right in red. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14–19 illustrates an alternative route for oxaloacetate produced in mitochondria. 8885d_c14_521-559 2/6/04 3:43 PM Page 544 mac76 mac76:385_reb: The reaction involves biotin as a carrier of activated HCO 3 H11002 (Fig. 14–18). The reaction mechanism is shown in Figure 16–16. Pyruvate carboxylase is the first regu- latory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. (Acetyl-CoA is pro- duced by fatty acid oxidation (Chapter 17), and its ac- cumulation signals the availability of fatty acids as fuel.) As we shall see in Chapter 16 (see Fig. 16–15), the pyru- vate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle. Because the mitochondrial membrane has no trans- porter for oxaloacetate, before export to the cytosol the oxaloacetate formed from pyruvate must be reduced to malate by mitochondrial malate dehydrogenase, at the expense of NADH: Oxaloacetate H11001 NADH H11001 H H11001 L-malate H11001 NAD H11001 (14–5) z y 14.4 Gluconeogenesis 545 TABLE 14–2 Free-Energy Changes of Glycolytic Reactions in Erythrocytes Glycolytic reaction step H9004GH11032H11034 (kJ/mol) H9004G (kJ/mol) 1 Glucose H11001 ATP 88n glucose 6-phosphate H11001 ADP H1100216.7 H1100233.4 2 Glucose 6-phosphate fructose 6-phosphate 1.7 0 to 25 3 Fructose 6-phosphate H11001 ATP On fructose 1,6-bisphosphate H11001 ADP H1100214.2 H1100222.2 4 Fructose 1,6-bisphosphate dihydroxyacetone phosphate H11001 glyceraldehyde 3-phosphate 23.8 0 to H110026 5 Dihydroxyacetone phosphate glyceraldehyde 3-phosphate 7.5 0 to 4 6 Glyceraldehyde 3-phosphate H11001 P i H11001 NAD H11001 1,3-bisphosphoglycerate H11001 NADH H11001 H H11001 6.3 H110022 to 2 7 1,3-Bisphosphoglycerate H11001 ADP 3-phosphoglycerate H11001 ATP H1100218.8 0 to 2 8 3-Phosphoglycerate 2-phosphoglycerate 4.4 0 to 0.8 9 2-Phosphoglycerate phosphoenolpyruvate H11001 H 2 O 7.5 0 to 3.3 10 Phosphoenolpyruvate H11001 ADP 88n pyruvate H11001 ATP H1100231.4 H1100216.7 z y z y z y z y z y z y z y Note: H9004GH11032H11034 is the standard free-energy change, as defined in Chapter 13 (p. 491). H9004G is the free-energy change calculated from the actual concentrations of glycolytic intermediates present under physiological conditions in erythrocytes, at pH 7. The glycolytic reactions bypassed in gluconeogenesis are shown in red. Biochemical equations are not necessarily balanced for H or charge (p. 506). PO 3 HO C H11002 O (b) C O C Oxaloacetate O O H11002 O H11002 C ATP C H11001 O Guanosine PEP carboxykinase Pyruvate biotin pyruvate carboxylase CH 3 ADP H11001 P i O Phosphoenolpyruvate O COO H11002 PO 2H11002 OO P O O H11002 P O O H11002 O H11002 O Bicarbonate O C O H11002 CO 2 H11001 GTP GDP CH 2 C O CH 2 O H11002 (a) Oxaloacetate FIGURE 14–17 Synthesis of phosphoenolpyruvate from pyruvate. (a) In mitochondria, pyruvate is converted to oxaloacetate in a biotin- requiring reaction catalyzed by pyruvate carboxylase. (b) In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxy- kinase. The CO 2 incorporated in the pyruvate carboxylase reaction is lost here as CO 2 . The decarboxylation leads to a rearrangement of electrons that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the H9253 phosphate of GTP. 8885d_c14_545 2/9/04 7:04 AM Page 545 mac76 mac76:385_reb: The standard free-energy change for this reaction is quite high, but under physiological conditions (includ- ing a very low concentration of oxaloacetate) H9004G ≈ 0 and the reaction is readily reversible. Mitochondrial malate dehydrogenase functions in both gluconeogenesis and the citric acid cycle, but the overall flow of metabolites in the two processes is in opposite directions. Malate leaves the mitochondrion through a specific transporter in the inner mitochondrial membrane (see Fig. 19–27), and in the cytosol it is reoxidized to ox- aloacetate, with the production of cytosolic NADH: Malate H11001 NAD H11001 88n oxaloacetate H11001 NADH H11001 H H11001 (14–6) The oxaloacetate is then converted to PEP by phosphoenolpyruvate carboxykinase (Fig. 14–17). This Mg 2H11001 -dependent reaction requires GTP as the phosphoryl group donor : Oxaloacetate H11001 GTP PEP H11001 CO 2 H11001 GDP (14–7) The reaction is reversible under intracellular conditions; the formation of one high-energy phosphate compound (PEP) is balanced by the hydrolysis of another (GTP). The overall equation for this set of bypass reactions, the sum of Equations 14–4 through 14–7, is Pyruvate H11001 ATP H11001 GTP H11001 HCO 3 H11002 88n PEP H11001 ADP H11001 GDP H11001 P i H11001 CO 2 H9004GH11032H11034 H11005 0.9 kJ/mol (14–8) Two high-energy phosphate equivalents (one from ATP and one from GTP), each yielding about 50 kJ/mol un- der cellular conditions, must be expended to phosphor- ylate one molecule of pyruvate to PEP. In contrast, when PEP is converted to pyruvate during glycolysis, only one ATP is generated from ADP. Although the standard free- energy change (H9004GH11032H11034) of the two-step path from pyru- vate to PEP is 0.9 kJ/mol, the actual free-energy change (H9004G), calculated from measured cellular concentrations of intermediates, is very strongly negative (H1100225 kJ/mol); this results from the ready consumption of PEP in other reactions such that its concentration remains relatively low. The reaction is thus effectively irreversible in the cell. Note that the CO 2 added to pyruvate in the pyru- vate carboxylase step is the same molecule that is lost in the PEP carboxykinase reaction (Fig. 14–17). This carboxylation-decarboxylation sequence represents a way of “activating” pyruvate, in that the decarboxyla- tion of oxaloacetate facilitates PEP formation. In Chap- ter 21 we shall see how a similar carboxylation-decar- boxylation sequence is used to activate acetyl-CoA for fatty acid biosynthesis (see Fig. 21–1). There is a logic to the route of these reactions through the mitochondrion. The [NADH]/[NAD H11001 ] ratio in the cytosol is 8 H11003 10 H110024 , about 10 5 times lower than in mitochondria. Because cytosolic NADH is consumed in gluconeogenesis (in the conversion of 1,3-bisphos- z y Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway546 O OP C S H11002 O O H11002 HN NH N H ATP Rib Adenine Enz H11002 O OH O OP O H11002 O C O H11002 O C H11002 O O H11002 O O C O O OP O H11002 C O O S N NH CH 2 H11002 H11002 O O C O C CH 2 H11002 O O H11002 O CC CH 2 N H Enz O O Bicarbonate Biotinyl-enzyme S HN NH N H Enz O O Biotinyl-enzyme ADP H11001 P i 1 2 Carboxybiotinyl-enzyme Pyruvate enolate H11001 Oxaloacetate FIGURE 14–18 Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to the enzyme through an amide linkage to the H9255-amino group of a Lys residue, forming a biotinyl-enzyme. The reaction occurs in two phases, which occur at two different sites in the enzyme. At catalytic site 1, bicarbonate ion is converted to CO 2 at the expense of ATP. Then CO 2 reacts with biotin, forming carboxybiotinyl-enzyme. The long arm composed of biotin and the side chain of the Lys to which it is attached then carry the CO 2 of carboxybiotinyl-enzyme to catalytic site 2 on the enzyme surface, where CO 2 is released and reacts with the pyruvate, forming oxaloacetate and regenerating the biotinyl-enzyme. The general role of flexible arms in carrying reaction intermediates between enzyme active sites is described in Figure 16–17, and the mechanistic details of the pyruvate carboxylase reaction are shown in Figure 16–16. Sim- ilar mechanisms occur in other biotin-dependent carboxylation reac- tions, such as those catalyzed by propionyl-CoA carboxylase (see Fig. 17–11) and acetyl-CoA carboxylase (see Fig. 21–1). 8885d_c14_546 2/9/04 7:05 AM Page 546 mac76 mac76:385_reb: phoglycerate to glyceraldehyde 3-phosphate; Fig. 14–16), glucose biosynthesis cannot proceed unless NADH is available. The transport of malate from the mi- tochondrion to the cytosol and its reconversion there to oxaloacetate effectively moves reducing equivalents to the cytosol, where they are scarce. This path from pyru- vate to PEP therefore provides an important balance be- tween NADH produced and consumed in the cytosol during gluconeogenesis. A second pyruvate n PEP bypass predominates when lactate is the glucogenic precursor (Fig. 14–19). This pathway makes use of lactate produced by glycol- ysis in erythrocytes or anaerobic muscle, for example, and it is particularly important in large vertebrates af- ter vigorous exercise (Box 14–1). The conversion of lac- tate to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria is therefore unnecessary. Af- ter the pyruvate produced by the lactate dehydrogenase reaction is transported into the mitochondrion, it is con- verted to oxaloacetate by pyruvate carboxylase, as de- scribed above. This oxaloacetate, however, is converted directly to PEP by a mitochondrial isozyme of PEP car- boxykinase, and the PEP is transported out of the mi- tochondrion to continue on the gluconeogenic path. The mitochondrial and cytosolic isozymes of PEP carboxy- kinase are encoded by separate genes in the nuclear chromosomes, providing another example of two dis- tinct enzymes catalyzing the same reaction but having different cellular locations or metabolic roles (recall the isozymes of hexokinase). Conversion of Fructose 1,6-Bisphosphate to Fructose 6-Phosphate Is the Second Bypass The second glycolytic reaction that cannot participate in gluconeogenesis is the phosphorylation of fructose 6- phosphate by PFK-1 (Table 14–2, step 3 ). Because this reaction is highly exergonic and therefore irreversible in intact cells, the generation of fructose 6-phosphate from fructose 1,6-bisphosphate (Fig. 14–16) is catalyzed by a different enzyme, Mg 2H11001 -dependent fructose 1,6- bisphosphatase (FBPase-1), which promotes the es- sentially irreversible hydrolysis of the C-1 phosphate (not phosphoryl group transfer to ADP): Fructose 1,6-bisphosphate H11001 H 2 O 88n fructose 6-phosphate H11001 P i H9004GH11032H11034 H11005 H1100216.3 kJ/mol Conversion of Glucose 6-Phosphate to Glucose Is the Third Bypass The third bypass is the final reaction of gluconeogene- sis, the dephosphorylation of glucose 6-phosphate to yield glucose (Fig. 14–16). Reversal of the hexokinase reaction (p. 526) would require phosphoryl group trans- fer from glucose 6-phosphate to ADP, forming ATP, an energetically unfavorable reaction (Table 14–2, step 1 ). The reaction catalyzed by glucose 6-phosphatase does not require synthesis of ATP; it is a simple hy- drolysis of a phosphate ester: Glucose 6-phosphate H11001 H 2 O On glucose H11001 P i H9004GH11032H11034 H11005 H1100213.8 kJ/mol This Mg 2H11001 -activated enzyme is found on the lumenal side of the endoplasmic reticulum of hepatocytes and renal cells (see Fig. 15–6). Muscle and brain tissue do not contain this enzyme and so cannot carry out gluco- neogenesis. Glucose produced by gluconeogenesis in the liver or kidney or ingested in the diet is delivered to brain and muscle through the bloodstream. 14.4 Gluconeogenesis 547 cytosolic malate dehydrogenase mitochondrial malate dehydrogenase Pyruvate Pyruvate Oxaloacetate Malate Malate Oxaloacetate cytosolic PEP carboxykinase CO 2 PEP CO 2 Oxaloacetate Pyruvate Lactate PEP mitochondrial PEP carboxykinase CO 2 pyruvate carboxylase NAD + lactate dehydrogenase Mitochondrion Cytosol Pyruvate pyruvate carboxylase NADH + H + NAD + NADH + H + NAD + NADH + H + CO 2 FIGURE 14–19 Alternative paths from pyruvate to phospho- enolpyruvate. The path that predominates depends on the glucogenic precursor (lactate or pyruvate). The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shut- tled out of the mitochondrion (see text). The relative importance of the two pathways depends on the availability of lactate and the cytosolic requirements for NADH by gluconeogenesis. 8885d_c14_521-559 2/6/04 3:43 PM Page 547 mac76 mac76:385_reb: Gluconeogenesis Is Energetically Expensive, but Essential The sum of the biosynthetic reactions leading from pyruvate to free blood glucose (Table 14–3) is 2 Pyruvate H11001 4ATP H11001 2GTP H11001 2NADH H11001 2H H11001 H11001 4H 2 O 88n glucose H11001 4ADP H11001 2GDP H11001 6P i H11001 2NAD H11001 (14–9) For each molecule of glucose formed from pyruvate, six high-energy phosphate groups are required, four from ATP and two from GTP. In addition, two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate. Clearly, Equation 14–9 is not simply the reverse of the equation for conversion of glucose to pyruvate by glycolysis, which requires only two molecules of ATP: Glucose H11001 2ADP H11001 2P i H11001 2NAD H11001 88n 2 pyruvate H11001 2ATP H11001 2NADH H11001 2H H11001 H11001 2H 2 O The synthesis of glucose from pyruvate is a relatively expensive process. Much of this high energy cost is nec- essary to ensure the irreversibility of gluconeogenesis. Under intracellular conditions, the overall free-energy change of glycolysis is at least H1100263 kJ/mol. Under the same conditions the overall H9004G of gluconeogenesis is H1100216 kJ/mol. Thus both glycolysis and gluconeogenesis are essentially irreversible processes in cells. Citric Acid Cycle Intermediates and Many Amino Acids Are Glucogenic The biosynthetic pathway to glucose described above allows the net synthesis of glucose not only from pyru- vate but also from the four-, five-, and six-carbon inter- mediates of the citric acid cycle (Chapter 16). Citrate, isocitrate, H9251-ketoglutarate, succinyl-CoA, succinate, fu- marate, and malate—all are citric acid cycle intermedi- ates that can undergo oxidation to oxaloacetate (see Fig. 16–7). Some or all of the carbon atoms of most amino acids derived from proteins are ultimately catab- olized to pyruvate or to intermediates of the citric acid cycle. Such amino acids can therefore undergo net con- version to glucose and are said to be glucogenic (Table 14–4). Alanine and glutamine, the principal molecules that transport amino groups from extrahepatic tissues to the liver (see Fig. 18–9), are particularly important glucogenic amino acids in mammals. After removal of their amino groups in liver mitochondria, the carbon skeletons remaining (pyruvate and H9251-ketoglutarate, re- spectively) are readily funneled into gluconeogenesis. In contrast, no net conversion of fatty acids to glu- cose occurs in mammals. As we shall see in Chapter 17, the catabolism of most fatty acids yields only acetyl- CoA. Mammals cannot use acetyl-CoA as a precursor of glucose, because the pyruvate dehydrogenase reaction is irreversible and cells have no other pathway to con- vert acetyl-CoA to pyruvate. Plants, yeast, and many bacteria do have a pathway (the glyoxylate cycle; see Fig. 16–20) for converting acetyl-CoA to oxaloacetate, so these organisms can use fatty acids as the starting material for gluconeogenesis. This is especially impor- tant during the germination of seedlings, before photo- synthesis can serve as a source of glucose. Glycolysis and Gluconeogenesis Are Regulated Reciprocally If glycolysis (the conversion of glucose to pyruvate) and gluconeogenesis (the conversion of pyruvate to glucose) were allowed to proceed simultaneously at high rates, Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway548 TABLE 14–3 Sequential Reactions in Gluconeogenesis Starting from Pyruvate Pyruvate H11001 HCO 3 H11002 H11001 ATP On oxaloacetate H11001 ADP H11001 P i H110032 Oxaloacetate H11001 GTP phosphoenolpyruvate H11001 CO 2 H11001 GDP H110032 Phosphoenolpyruvate H11001 H 2 O 2-phosphoglycerate H110032 2-Phosphoglycerate 3-phosphoglycerate H110032 3-Phosphoglycerate H11001 ATP 1,3-bisphosphoglycerate H11001 ADP H110032 1,3-Bisphosphoglycerate H11001 NADH H11001 H H11001 glyceraldehyde 3-phosphate H11001 NAD H11001 H11001 P i H110032 Glyceraldehyde 3-phosphate dihydroxyacetone phosphate Glyceraldehyde 3-phosphate H11001 dihydroxyacetone phosphate fructose 1,6-bisphosphate Fructose 1,6-bisphosphate On fructose 6-phosphate H11001 P i Fructose 6-phosphate glucose 6-phosphate Glucose 6-phosphate H11001 H 2 O On glucose H11001 P i Sum: 2 Pyruvate H11001 4ATP H11001 2GTP H11001 2NADH H11001 2H H11001 H11001 4H 2 O On glucose H11001 4ADP H11001 2GDP H11001 6P i H11001 2NAD H11001 z y z y z y z y z y z y z y z y Note: The bypass reactions are in red; all other reactions are reversible steps of glycolysis. The figures at the right indicate that the reaction is to be counted twice, because two three-carbon precursors are required to make a molecule of glucose. The reactions required to replace the cytosolic NADH consumed in the glycer- aldehyde 3-phosphate dehydrogenase reaction (the conversion of lactate to pyruvate in the cytosol or the transport of reducing equivalents from mitochondria to the cytosol in the form of malate) are not considered in this summary. Biochemical equations are not necessarily balanced for H and charge (p. 506). 8885d_c14_521-559 2/6/04 3:43 PM Page 548 mac76 mac76:385_reb: the result would be the consumption of ATP and the production of heat. For example, PFK-1 and FBPase-1 catalyze opposing reactions: ATP H11001 fructose 6-phosphate 8888888n PFK–1 ADP H11001 fructose 1,6-bisphosphate Fructose 1,6-bisphosphate H11001 H 2 O 8888888n FBPase–1 fructose 6-phosphate H11001 P i The sum of these two reactions is ATP H11001 H 2 O 88n ADP H11001 P i H11001 heat These two enzymatic reactions, and a number of others in the two pathways, are regulated allosterically and by covalent modification (phosphorylation). In Chapter 15 we take up the mechanisms of this regulation in detail. For now, suffice it to say that the pathways are regu- lated so that when the flux of glucose through glycoly- sis goes up, the flux of pyruvate toward glucose goes down, and vice versa. SUMMARY 14.4 Gluconeogenesis ■ Gluconeogenesis is a ubiquitous multistep process in which pyruvate or a related three-carbon compound (lactate, alanine) is converted to glucose. Seven of the steps in gluconeogenesis are catalyzed by the same enzymes used in glycolysis; these are the reversible reactions. ■ Three irreversible steps in the glycolytic pathway are bypassed by reactions catalyzed by gluconeogenic enzymes: (1) conversion of pyruvate to PEP via oxaloacetate, catalyzed by pyruvate carboxylase and PEP carboxykinase; (2) dephosphorylation of fructose 1,6-bisphosphate by FBPase-1; and (3) dephosphorylation of glucose 6-phosphate by glucose 6-phosphatase. ■ Formation of one molecule of glucose from pyruvate requires 4 ATP, 2 GTP, and 2 NADH; it is expensive. ■ In mammals, gluconeogenesis in the liver and kidney provides glucose for use by the brain, muscles, and erythrocytes. ■ Pyruvate carboxylase is stimulated by acetyl-CoA, increasing the rate of gluconeogenesis when the cell already has adequate supplies of other substrates (fatty acids) for energy production. ■ Animals cannot convert acetyl-CoA derived from fatty acids into glucose; plants and microorganisms can. ■ Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful operation of both pathways at the same time. 14.5 Pentose Phosphate Pathway of Glucose Oxidation In most animal tissues, the major catabolic fate of glucose 6-phosphate is glycolytic breakdown to pyruvate, much of which is then oxidized via the citric acid cycle, ultimately leading to the formation of ATP. Glucose 6-phosphate does have other catabolic fates, however, which lead to specialized products needed by the cell. Of particular importance in some tissues is the oxidation of glucose 6-phosphate to pen- tose phosphates by the pentose phosphate pathway (also called the phosphogluconate pathway or the hexose monophosphate pathway; Fig. 14–20). In this oxidative pathway, NADP H11001 is the electron acceptor, yielding NADPH. Rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa, use the pen- toses to make RNA, DNA, and such coenzymes as ATP, NADH, FADH 2 , and coenzyme A. In other tissues, the essential product of the pen- tose phosphate pathway is not the pentoses but the elec- tron donor NADPH, needed for reductive biosynthesis or to counter the damaging effects of oxygen radicals. Tissues that carry out extensive fatty acid synthesis (liver, adipose, lactating mammary gland) or very ac- tive synthesis of cholesterol and steroid hormones (liver, adrenal gland, gonads) require the NADPH pro- vided by the pathway. Erythrocytes and the cells of the lens and cornea are directly exposed to oxygen and thus to the damaging free radicals generated by oxygen. 14.5 Pentose Phosphate Pathway of Glucose Oxidation 549 Pyruvate Alanine Cysteine Glycine Serine Threonine Tryptophan* H9251-Ketoglutarate Arginine Glutamate Glutamine Histidine Proline Glucogenic Amino Acids, Grouped by Site of Entry Note: All these amino acids are precursors of blood glucose or liver glycogen, because they can be converted to pyruvate or citric acid cycle intermediates. Of the 20 common amino acids, only leucine and lysine are unable to furnish carbon for net glucose synthesis. *These amino acids are also ketogenic (see Fig. 18–21). TABLE 14–4 Succinyl-CoA Isoleucine* Methionine Threonine Valine Fumarate Phenylalanine* Tyrosine* Oxaloacetate Asparagine Aspartate 8885d_c14_521-559 2/6/04 3:43 PM Page 549 mac76 mac76:385_reb: By maintaining a reducing atmosphere (a high ratio of NADPH to NADP H11001 and a high ratio of reduced to oxi- dized glutathione), they can prevent or undo oxidative damage to proteins, lipids, and other sensitive molecules. In erythrocytes, the NADPH produced by the pentose phosphate pathway is so important in preventing oxida- tive damage that a genetic defect in glucose 6-phosphate dehydrogenase, the first enzyme of the pathway, can have serious medical consequences (Box 14–3). ■ The Oxidative Phase Produces Pentose Phosphates and NADPH The first reaction of the pentose phosphate pathway (Fig. 14–21) is the oxidation of glucose 6-phosphate by glucose 6-phosphate dehydrogenase (G6PD) to form 6-phosphoglucono-H9254-lactone, an intramolecular ester. NADP H11001 is the electron acceptor, and the overall equilibrium lies far in the direction of NADPH forma- tion. The lactone is hydrolyzed to the free acid 6-phos- phogluconate by a specific lactonase, then 6-phospho- gluconate undergoes oxidation and decarboxylation by 6-phosphogluconate dehydrogenase to form the ke- topentose ribulose 5-phosphate. This reaction generates Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway550 Nonoxidative phase Oxidative phase Glucose 6-phosphate 6-Phosphogluconate CO 2 Ribulose 5-phosphate Ribose 5-phosphate Nucleotides, coenzymes, DNA, RNA NADP H11001 NADPH 2 GSH GSSG Fatty acids, sterols, etc. Precursors transketolase, transaldolase glutathione reductase reductive biosynthesis NADP H11001 NADPH HOCH O CH 2 OH H11001 H H11001 lactonase A DM O H11002 C HCOH A 6-Phospho- gluconate Glucose 6-phosphate D-Ribose 5-phosphate phosphopentose isomerase glucose 6-phosphate dehydrogenase 6-phosphogluconate dehydrogenase A HC A O 3 A HCOH C HCOH HOCH P A HCOH A HCOH O A CO 2 3 A D-Ribulose 5-phosphate Mg 2H11001 Mg 2H11001 6-Phospho- glucono-H9254-lactone NADP H11001 NADPH A HC O A HCOH A A HCOH A HCOH A H11001 H H11001 NADP H11001 2H11002 CH 2 OPO 2H11002 OCP HOCH A HCOH A A HCOH A A HCOH CHO A HCOH A A HCOH NADPH CH 2 OPO 3 2H11002 CH 2 OPO 3 2H11002 H 2 O 3 2H11002 CH 2 OPO Mg 2H11001 CH 2 OPO FIGURE 14–20 General scheme of the pentose phosphate pathway. NADPH formed in the oxidative phase is used to reduce glutathione, GSSG (see Box 14–3) and to support reductive biosynthesis. The other product of the oxidative phase is ribose 5-phosphate, which serves as precursor for nucleotides, coenzymes, and nucleic acids. In cells that are not using ribose 5-phosphate for biosynthesis, the nonoxidative phase recycles six molecules of the pentose into five molecules of the hexose glucose 6-phosphate, allowing continued production of NADPH and converting glucose 6-phosphate (in six cycles) to CO 2 . FIGURE 14–21 Oxidative reactions of the pentose phosphate path- way. The end products are ribose 5-phosphate, CO 2 , and NADPH. 8885d_c14_521-559 2/6/04 3:43 PM Page 550 mac76 mac76:385_reb: 14.5 Pentose Phosphate Pathway of Glucose Oxidation 551 BOX 14–3 BIOCHEMISTRY IN MEDICINE Why Pythagoras Wouldn’t Eat Falafel: Glucose 6-Phosphate Dehydrogenase Deficiency Fava beans, an ingredient of falafel, have been an im- portant food source in the Mediterranean and Middle East since antiquity. The Greek philosopher and math- ematician Pythagoras prohibited his followers from dining on fava beans, perhaps because they make many people sick with a condition called favism, which can be fatal. In favism, erythrocytes begin to lyse 24 to 48 hours after ingestion of the beans, releasing free hemoglobin into the blood. Jaundice and sometimes kidney failure can result. Similar symptoms can occur with ingestion of the antimalarial drug primaquine or of sulfa antibiotics or following exposure to certain herbicides. These symptoms have a genetic basis: glu- cose 6-phosphate dehydrogenase (G6PD) deficiency, which affects about 400 million people. Most G6PD- deficient individuals are asymptomatic; only the com- bination of G6PD deficiency and certain environmen- tal factors produces the clinical manifestations. G6PD catalyzes the first step in the pentose phos- phate pathway (see Fig. 14–21), which produces NADPH. This reductant, essential in many biosyn- thetic pathways, also protects cells from oxidative damage by hydrogen peroxide (H 2 O 2 ) and superoxide free radicals, highly reactive oxidants generated as metabolic byproducts and through the actions of drugs such as primaquine and natural products such as di- vicine—the toxic ingredient of fava beans. During normal detoxification, H 2 O 2 is converted to H 2 O by re- duced glutathione and glutathione peroxidase, and the oxidized glutathione is converted back to the reduced form by glutathione reductase and NADPH (Fig. 1). H 2 O 2 is also broken down to H 2 O and O 2 by catalase, which also requires NADPH. In G6PD-deficient individuals, the NADPH production is diminished and detoxification of H 2 O 2 is inhibited. Cellular damage results: lipid peroxidation leading to breakdown of erythrocyte membranes and oxidation of proteins and DNA. The geographic distribution of G6PD deficiency is instructive. Frequencies as high as 25% occur in trop- ical Africa, parts of the Middle East, and Southeast Asia, areas where malaria is most prevalent. In addi- tion to such epidemiological observations, in vitro studies show that growth of one malaria parasite, Plas- modium falciparum, is inhibited in G6PD-deficient erythrocytes. The parasite is very sensitive to oxida- tive damage and is killed by a level of oxidative stress that is tolerable to a G6PD-deficient human host. Be- cause the advantage of resistance to malaria balances the disadvantage of lowered resistance to oxidative damage, natural selection sustains the G6PD-deficient genotype in human populations where malaria is prevalent. Only under overwhelming oxidative stress, caused by drugs, herbicides, or divicine, does G6PD deficiency cause serious medical problems. An antimalarial drug such as primaquine is be- lieved to act by causing oxidative stress to the para- site. It is ironic that antimalarial drugs can cause ill- ness through the same biochemical mechanism that provides resistance to malaria. Divicine also acts as an antimalarial drug, and ingestion of fava beans may pro- tect against malaria. By refusing to eat falafel, many Pythagoreans with normal G6PD activity may have un- wittingly increased their risk of malaria! FIGURE 1 Role of NADPH and glutathione in protecting cells against highly reactive oxygen derivatives. Reduced glutathione (GSH) protects the cell by destroying hydrogen peroxide and hy- droxyl free radicals. Regeneration of GSH from its oxidized form (GSSG) requires the NADPH produced in the glucose 6-phosphate dehydrogenase reaction. Mitochondrial respiration, ionizing radiation, sulfa drugs, herbicides, antimalarials, divicine Oxidative damage to lipids, proteins, DNA O 2 H11002 O 2 2H H11001 H H11001 e H11002 OH H 2 O 2 H 2 O 2H 2 O Superoxide radical Hydrogen peroxide Hydroxyl free radical NADP H11001 NADPH H H11001 H11001 Glucose 6-phosphate 2GSH GSSG 6-Phospho- glucono-d-lactone glucose 6-phosphate dehydrogenase (G6PD) glutathione reductase glutathione peroxidase e H11002 8885d_c14_521-559 2/6/04 3:43 PM Page 551 mac76 mac76:385_reb: a second molecule of NADPH. Phosphopentose iso- merase converts ribulose 5-phosphate to its aldose iso- mer, ribose 5-phosphate. In some tissues, the pentose phosphate pathway ends at this point, and its overall equation is Glucose 6-phosphate H11001 2NADP H11001 H11001 H 2 O 88n ribose 5-phosphate H11001 CO 2 H11001 2NADPH H11001 2H H11001 The net result is the production of NADPH, a reductant for biosynthetic reactions, and ribose 5-phosphate, a precursor for nucleotide synthesis. The Nonoxidative Phase Recycles Pentose Phosphates to Glucose 6-Phosphate In tissues that require primarily NADPH, the pentose phosphates produced in the oxidative phase of the path- way are recycled into glucose 6-phosphate. In this non- oxidative phase, ribulose 5-phosphate is first epimerized to xylulose 5-phosphate: Then, in a series of rearrangements of the carbon skele- tons (Fig. 14–22), six five-carbon sugar phosphates are CH 2 OH OC OHH OHH C C CH 2 OPO 3 2H11002 CH 2 OH OC HO H OHH C C CH 2 OPO 3 2H11002 ribose 5-phosphate epimerase Ribulose 5-phosphate Xylulose 5-phosphate converted to five six-carbon sugar phosphates, com- pleting the cycle and allowing continued oxidation of glucose 6-phosphate with production of NADPH. Con- tinued recycling leads ultimately to the conversion of glucose 6-phosphate to six CO 2 . Two enzymes unique to the pentose phosphate pathway act in these intercon- versions of sugars: transketolase and transaldolase. Transketolase catalyzes the transfer of a two-carbon fragment from a ketose donor to an aldose acceptor (Fig. 14–23a). In its first appearance in the pentose phosphate pathway, transketolase transfers C-1 and C-2 of xylulose 5-phosphate to ribose 5-phosphate, forming the seven-carbon product sedoheptulose 7-phosphate (Fig. 14–23b). The remaining three-carbon fragment from xylulose is glyceraldehyde 3-phosphate. Next, transaldolase catalyzes a reaction similar to the aldolase reaction of glycolysis: a three-carbon frag- ment is removed from sedoheptulose 7-phosphate and condensed with glyceraldehyde 3-phosphate, forming fructose 6-phosphate and the tetrose erythrose 4-phos- phate (Fig. 14–24). Now transketolase acts again, form- ing fructose 6-phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate (Fig. 14–25). Two molecules of glyceraldehyde 3-phos- phate formed by two iterations of these reactions can be converted to a molecule of fructose 1,6-bisphosphate as in gluconeogenesis (Fig. 14–16), and finally FBPase-1 and phosphohexose isomerase convert fructose 1,6-bisphos- phate to glucose 6-phosphate. The cycle is complete: six pentose phosphates have been converted to five hexose phosphates (Fig. 14–22b). Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway552 Sedoheptulose 7-phosphate fructose 1,6- bisphosphatase Glyceraldehyde 3-phosphate Fructose 6-phosphate Erythrose 4-phosphate Xylulose 5-phosphate transketolase transaldolase transketolase Xylulose 5-phosphate Glyceraldehyde 3-phosphate Fructose 6-phosphate Glucose 6-phosphate aldolase triose phosphate isomerase Ribose 5-phosphate epimerase (a) phosphohexose isomerase oxidative reactions of pentose phosphate pathway 6C 6C 6C 6C (b) 7C5C 5C 4C 3C 6C 3C 5C 4C 3C 7C 3C 5C 5C 5C FIGURE 14–22 Nonoxidative reactions of the pentose phosphate pathway. (a) These reactions convert pentose phosphates to hexose phosphates, allowing the oxidative reactions (see Fig. 14–21) to con- tinue. The enzymes transketolase and transaldolase are specific to this pathway; the other enzymes also serve in the glycolytic or gluco- neogenic pathways. (b) A schematic diagram showing the pathway from six pentoses (5C) to five hexoses (6C). Note that this involves two sets of the interconversions shown in (a). Every reaction shown here is reversible; unidirectional arrows are used only to make clear the direction of the reactions during continuous oxidation of glucose 6- phosphate. In the light-independent reactions of photosynthesis, the direction of these reactions is reversed (see Fig. 20–10). 8885d_c14_521-559 2/6/04 3:43 PM Page 552 mac76 mac76:385_reb: 14.5 Pentose Phosphate Pathway of Glucose Oxidation 553 CO CHOH H11001 Ketose donor Aldose acceptor TPP transketolase (a) CH 2 OH R 2 C O R 1 CHOH R 2 R 1 CH 2 OH C O O C H11001 H H H11001 Xylulose 5-phosphate Ribose 5-phosphate Glyceraldehyde 3-phosphate Sedoheptulose 7-phosphate TPP transketolase (b) C O CH 2 OH C O O C C H11001 H HOH CHOH C CH 2 OPO 3 2H11002 HOH H CHOH CHOH C CH 2 OPO 3 2H11002 HOHC CH 2 OPO 3 2H11002 HOH CHO H C CH 2 OPO 3 2H11002 CH 2 OH C O CHO H HOH FIGURE 14–23 The first reaction catalyzed by transketolase. (a) The general reaction catalyzed by trans- ketolase is the transfer of a two- carbon group, carried temporarily on enzyme-bound TPP, from a ketose donor to an aldose acceptor. (b) Conversion of two pentose phosphates to a triose phosphate and a seven-carbon sugar phosphate, sedoheptulose 7-phosphate. H11001 Glyceraldehyde 3-phosphate Erythrose 4-phosphate Fructose 6-phosphate transaldolase O C H11001 H C CH 2 OPO 3 2H11002 HOH CHOH C CH 2 OPO 3 2H11002 HOHC CH 2 OPO 3 2H11002 HOH C O H CHOH CHO H Sedoheptulose 7-phosphate CHOH CHOH C CH 2 OPO 3 2H11002 HOH CHO H CH 2 OH C O CH 2 OH C O FIGURE 14–24 The reaction catalyzed by transaldolase. H11001 Glyceraldehyde 3-phosphate Erythrose 4-phosphate Fructose 6-phosphate transketolase H11001 C CH 2 OPO 3 2H11002 HOH O C H CHOH CHOH C CH 2 OPO 3 2H11002 HOHC CH 2 OPO 3 2H11002 HOH C O H CHO H Xylulose 5-phosphate CHOH CH 2 OPO 3 2H11002 CHO H CH 2 OH C O CH 2 OH C O TPP FIGURE 14–25 The second reaction catalyzed by transketolase. Transketolase requires the cofactor thiamine py- rophosphate (TPP), which stabilizes a two-carbon car- banion in this reaction (Fig. 14–26a), just as it does in the pyruvate decarboxylase reaction (Fig. 14–13). Transaldolase uses a Lys side chain to form a Schiff base with the carbonyl group of its substrate, a ketose, thereby stabilizing a carbanion (Fig. 14–26b) that is cen- tral to the reaction mechanism. The process described in Figure 14–21 is known as the oxidative pentose phosphate pathway. The first two steps are oxidations with large, negative standard free-energy changes and are essentially irreversible in 8885d_c14_521-559 2/6/04 3:43 PM Page 553 mac76 mac76:385_reb: the cell. The reactions of the nonoxidative part of the pentose phosphate pathway (Fig. 14–22) are readily re- versible and thus also provide a means of converting hexose phosphates to pentose phosphates. As we shall see in Chapter 20, a process that converts hexose phos- phates to pentose phosphates is crucial to the photo- synthetic assimilation of CO 2 by plants. That pathway, the reductive pentose phosphate pathway, is es- sentially the reversal of the reactions shown in Figure 14–22 and employs many of the same enzymes. All the enzymes in the pentose phosphate pathway are located in the cytosol, like those of glycolysis and most of those of gluconeogenesis. In fact, these three pathways are connected through several shared inter- mediates and enzymes. The glyceraldehyde 3-phos- phate formed by the action of transketolase is readily converted to dihydroxyacetone phosphate by the gly- colytic enzyme triose phosphate isomerase, and these two trioses can be joined by the aldolase as in gluco- neogenesis, forming fructose 1,6-bisphosphate. Alterna- tively, the triose phosphates can be oxidized to pyru- vate by the glycolytic reactions. The fate of the trioses is determined by the cell’s relative needs for pentose phosphates, NADPH, and ATP. Wernicke-Korsakoff Syndrome Is Exacerbated by a Defect in Transketolase In humans with Wernicke-Korsakoff syndrome, a mutation in the gene for transketolase results in an enzyme having an affinity for its coenzyme TPP that is one-tenth that of the normal enzyme. Although mod- erate deficiencies in the vitamin thiamine have little ef- fect on individuals with an unmutated transketolase gene, in those with the altered gene, thiamine deficiency drops the level of TPP below that needed to saturate the enzyme. The lowering of transketolase activity slows the whole pentose phosphate pathway, and the result is the Wernicke-Korsakoff syndrome: severe memory loss, mental confusion, and partial paralysis. The syndrome is more common among alcoholics than in the general population; chronic alcohol consumption interferes with the intestinal absorption of some vitamins, including thiamine. ■ Glucose 6-Phosphate Is Partitioned between Glycolysis and the Pentose Phosphate Pathway Whether glucose 6-phosphate enters glycolysis or the pentose phosphate pathway depends on the current needs of the cell and on the concentration of NADP H11001 in the cytosol. Without this electron acceptor, the first reaction of the pentose phosphate pathway (catalyzed by G6PD) cannot proceed. When a cell is rapidly con- verting NADPH to NADP H11001 in biosynthetic reductions, the level of NADP H11001 rises, allosterically stimulating G6PD and thereby increasing the flux of glucose 6-phosphate through the pentose phosphate pathway (Fig. 14–27). When the demand for NADPH slows, the level of NADP H11001 drops, the pentose phosphate pathway slows, and glucose 6-phosphate is instead used to fuel glycolysis. Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway554 Glucose Glucose 6-phosphate pentose phosphate pathway glycolysis 6-Phospho- gluconolactone Pentose phosphates ATP NADPH NADPH (a) Transketolase (b) Transaldolase OH C H11002 C H11002 HOH 2 C C C C C RN N H S TPP H RH11032 CH 3 CH 2 OH 1 2 3 4 5 OH HOH 2 C C RN S RH11032 CH 3 resonance stabilization resonance stabilization H11001 H11001 Lys Lys OH C N H H CH 2 OH OH Protonated Schiff base FIGURE 14–26 Carbanion intermediates stabilized by covalent in- teractions with transketolase and transaldolase. (a) The ring of TPP stabilizes the two-carbon carbanion carried by transketolase; see Fig. 14–13 for the chemistry of TPP action. (b) In the transaldolase reac- tion, the protonated Schiff base formed between the H9255-amino group of a Lys side chain and the substrate stabilizes a three-carbon carbanion. FIGURE 14–27 Role of NADPH in regulating the partitioning of glu- cose 6-phosphate between glycolysis and the pentose phosphate pathway. When NADPH is forming faster than it is being used for biosynthesis and glutathione reduction (see Fig. 14–20), [NADPH] rises and inhibits the first enzyme in the pentose phosphate pathway. As a result, more glucose 6-phosphate is available for glycolysis. 8885d_c14_521-559 2/6/04 3:43 PM Page 554 mac76 mac76:385_reb: SUMMARY 14.5 Pentose Phosphate Pathway of Glucose Oxidation ■ The oxidative pentose phosphate pathway (phosphogluconate pathway, or hexose monophosphate pathway) brings about oxidation and decarboxylation at C-1 of glucose 6-phosphate, reducing NADP H11001 to NADPH and producing pentose phosphates. ■ NADPH provides reducing power for biosynthetic reactions, and ribose 5-phosphate is a precursor for nucleotide and nucleic acid synthesis. Rapidly growing tissues and tissues carrying out active biosynthesis of fatty acids, cholesterol, or steroid hormones send more glucose 6-phosphate through the pentose phosphate pathway than do tissues with less demand for pentose phosphates and reducing power. ■ The first phase of the pentose phosphate pathway consists of two oxidations that convert glucose 6-phosphate to ribulose 5-phosphate and reduce NADP H11001 to NADPH. The second phase comprises nonoxidative steps that convert pentose phosphates to glucose 6-phosphate, which begins the cycle again. ■ In the second phase, transaldolase (with TPP as cofactor) and transketolase catalyze the interconversion of three-, four-, five-, six-, and seven-carbon sugars, with the reversible conversion of six pentose phosphates to five hexose phosphates. In the carbon-assimilating reactions of photosynthesis, the same enzymes catalyze the reverse process, called the reductive pentose phosphate pathway: conversion of five hexose phosphates to six pentose phosphates. ■ A genetic defect in transketolase that lowers its affinity for TPP exacerbates the Wernicke- Korsakoff syndrome. ■ Entry of glucose 6-phosphate either into glycolysis or into the pentose phosphate pathway is largely determined by the relative concentrations of NADP H11001 and NADPH. Chapter 14 Further Reading 555 Terms in bold are defined in the glossary. Key Terms glycolysis 522 fermentation 522 lactic acid fermentation hypoxia 523 ethanol (alcohol) fermentation 523 isozymes 526 acyl phosphate 530 substrate-level phos- phorylation 531 respiration-linked phos- phorylation 531 phosphoenolpyruvate (PEP) 532 mutases 534 isomerases 534 lactose intolerance galactosemia 537 thiamine pyrophos- phate (TPP) 540 gluconeogenesis 543 biotin 544 pentose phosphate pathway 549 phosphogluconate pathway 549 hexose monophosphate pathway 549 Further Reading General Fruton, J.S. (1999) Proteins, Genes, and Enzymes: The Inter- play of Chemistry and Biology, Yale University Press, New Haven. This text includes a detailed historical account of research on glycolysis. Glycolysis Boiteux, A. & Hess, B. (1981) Design of glycolysis. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 293, 5–22. Intermediate-level review of the pathway and the classic view of its control. Dandekar, T., Schuster, S., Snel, B., Huynen, M., & Bork, P. (1999) Pathway alignment: application to the comparative analysis of glycolytic enzymes. Biochem. J. 343, 115–124. Intermediate-level review of the bioinformatic view of the evo- lution of glycolysis. Dang, C.V. & Semenza, G.L. (1999) Oncogenic alterations of me- tabolism. Trends Biochem. Sci. 24, 68–72. Brief review of the molecular basis for increased glycolysis in tumors. Erlandsen, H., Abola, E.E., & Stevens, R.C. (2000) Combining structural genomics and enzymology: completing the picture in metabolic pathways and enzyme active sites. Curr. Opin. Struct. Biol. 10, 719–730. Intermediate-level review of the structures of the glycolytic enzymes. Hardie, D.G. (2000) Metabolic control: a new solution to an old problem. Curr. Biol. 10, R757–R759. Harris, A.L. (2002) Hypoxia—a key regulatory factor in tumour growth. Nat. Rev. Cancer 2, 38–47. 8885d_c14_521-559 2/6/04 3:43 PM Page 555 mac76 mac76:385_reb: Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway556 Heinrich, R., Melendez-Hevia, E., Montero, F., Nuno, J.C., Stephani, A., & Waddell, T.D. (1999) The structural design of glycolysis: an evolutionary approach. Biochem. Soc. Trans. 27, 294–298. Knowles, J. & Albery, W.J. (1977) Perfection in enzyme cataly- sis: the energetics of triose phosphate isomerase. Acc. Chem. Res. 10, 105–111. Phillips, D., Blake, C.C.F., & Watson, H.C. (eds) (1981) The Enzymes of Glycolysis: Structure, Activity and Evolution. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 293, 1–214. A collection of excellent reviews on the enzymes of glycolysis, written at a level challenging but comprehensible to a begin- ning student of biochemistry. Plaxton, W.C. (1996) The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 185–214. Very helpful review of the subcellular localization of glycolytic enzymes and the regulation of glycolysis in plants. Rose, I. (1981) Chemistry of proton abstraction by glycolytic en- zymes (aldolase, isomerases, and pyruvate kinase). Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 293, 131–144. Intermediate-level review of the mechanisms of these enzymes. Shirmer, T. & Evans, P.R. (1990) Structural basis for the al- losteric behavior of phosphofructokinase. Nature 343, 140–145. Smith, T.A. (2000) Mammalian hexokinases and their abnormal expression in cancer. Br. J. Biomed. Sci. 57, 170–178. A review of the four hexokinase isozymes of mammals: their properties and tissue distributions and their expression during the development of tumors. Feeder Pathways for Glycolysis Elsas, L.J. & Lai, K. (1998) The molecular biology of galac- tosemia. Genet. Med. 1, 40–48. Novelli, G. & Reichardt, J.K. (2000) Molecular basis of disor- ders of human galactose metabolism: past, present, and future. Mol. Genet. Metab. 71, 62–65. Petry, K.G. & Reichardt, J.K. (1998) The fundamental impor- tance of human galactose metabolism: lessons from genetics and biochemistry. Trends Genet. 14, 98–102. Van Beers, E.H., Buller, H.A., Grand, R.J., Einerhand, A.W.C., & Dekker, J. (1995) Intestinal brush border glycohydro- lases: structure, function, and development. Crit. Rev. Biochem. Mol. Biol. 30, 197–262. Fermentations Behal, R.H., Buxton, D.B., Robertson, J.G., & Olson, M.S. (1993) Regulation of the pyruvate dehydrogenase multienzyme complex. Annu. Rev. Nutr. 13, 497–520. Patel, M.S., Naik, S., Wexler, I.D., & Kerr, D.S. (1995) Gene regulation and genetic defects in the pyruvate dehydrogenase com- plex. J. Nutr. 125, 1753S–1757S. Patel, M.S. & Roche, T.E. (1990) Molecular biology and bio- chemistry of pyruvate dehydrogenase complexes. FASEB J. 4, 3224–3233. Robinson, B.H., MacKay, N., Chun, K., & Ling, M. (1996) Dis- orders of pyruvate carboxylase and the pyruvate dehydrogenase complex. J. Inherit. Metab. Dis. 19, 452–462. Gluconeogenesis Gerich, J.E., Meyer, C., Woerle, H.J., & Stumvoll, M. (2001) Renal gluconeogenesis: its importance in human glucose homeosta- sis. Diabetes Care 24, 382–391. Intermediate-level review of the contribution of kidney tissue to gluconeogenesis. Gleeson, T. (1996) Post-exercise lactate metabolism: a compara- tive review of sites, pathways, and regulation. Annu. Rev. Physiol. 58, 565–581. Hers, H.G. & Hue, L. (1983) Gluconeogenesis and related as- pects of glycolysis. Annu. Rev. Biochem. 52, 617–653. Matte, A., Tari, L.W., Goldie, H., & Delbaere, L.T.J. (1997) Structure and mechanism of phosphoenolpyruvate carboxykinase. J. Biol. Chem. 272, 8105–8108. Oxidative Pentose Phosphate Pathway Chayen, J., Howat, D.W., & Bitensky, L. (1986) Cellular bio- chemistry of glucose 6-phosphate and 6-phosphogluconate dehy- drogenase activities. Cell Biochem. Funct. 4, 249–253. Horecker, B.L. (1976) Unraveling the pentose phosphate path- way. In Reflections on Biochemistry (Kornberg, A., Cornudella, L., Horecker, B.L., & Oro, J., eds), pp. 65–72, Pergamon Press, Inc., Oxford. Kletzien, R.F., Harris, P.K., & Foellmi, L.A. (1994) Glucose 6-phosphate dehydrogenase: a “housekeeping” enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress. FASEB J. 8, 174–181. An intermediate-level review. Luzzato, L., Mehta, A., & Vulliamy, T. (2001) Glucose 6-phos- phate dehydrogenase deficiency. In The Metabolic and Molecular Bases of Inherited Disease, 8th edn (Scriver, C.R., Sly, W.S., Childs, B., Beaudet, A.L., Valle, D., Kinzler, K.W., & Vogelstein, B., eds), pp. 4517–4553, McGraw-Hill Inc., New York. The four-volume treatise in which this article appears is filled with fascinating information about the clinical and biochemical features of hundreds of inherited diseases of metabolism. Martini, G. & Ursini, M.V. (1996) A new lease on life for an old enzyme. BioEssays 18, 631–637. An intermediate-level review of glucose 6-phosphate dehydro- genase, the effects of mutations in this enzyme in humans, and the effects of knock-out mutations in mice. Notaro, R., Afolayan, A., & Luzzatto, L. (2000) Human muta- tions in glucose 6-phosphate dehydrogenase reflect evolutionary history. FASEB J. 14, 485–494. Wood, T. (1985) The Pentose Phosphate Pathway, Academic Press, Inc., Orlando, FL. Wood, T. (1986) Physiological functions of the pentose phosphate pathway. Cell Biochem. Funct. 4, 241–247. 8885d_c14_521-559 2/6/04 3:43 PM Page 556 mac76 mac76:385_reb: Chapter 14 Problems 557 1. Equation for the Preparatory Phase of Glycolysis Write balanced biochemical equations for all the reactions in the catabolism of glucose to two molecules of glyceraldehyde 3-phosphate (the preparatory phase of glycolysis), including the standard free-energy change for each reaction. Then write the overall or net equation for the preparatory phase of gly- colysis, with the net standard free-energy change. 2. The Payoff Phase of Glycolysis in Skeletal Muscle In working skeletal muscle under anaerobic conditions, glyc- eraldehyde 3-phosphate is converted to pyruvate (the payoff phase of glycolysis), and the pyruvate is reduced to lactate. Write balanced biochemical equations for all the reactions in this process, with the standard free-energy change for each reaction. Then write the overall or net equation for the pay- off phase of glycolysis (with lactate as the end product), in- cluding the net standard free-energy change. 3. Pathway of Atoms in Fermentation A “pulse-chase” experiment using 14 C-labeled carbon sources is carried out on a yeast extract maintained under strictly anaerobic con- ditions to produce ethanol. The experiment consists of incu- bating a small amount of 14 C-labeled substrate (the pulse) with the yeast extract just long enough for each intermedi- ate in the fermentation pathway to become labeled. The la- bel is then “chased” through the pathway by the addition of excess unlabeled glucose. The chase effectively prevents any further entry of labeled glucose into the pathway. (a) If [1- 14 C]glucose (glucose labeled at C-1 with 14 C) is used as a substrate, what is the location of 14 C in the prod- uct ethanol? Explain. (b) Where would 14 C have to be located in the starting glucose to ensure that all the 14 C activity is liberated as 14 CO 2 during fermentation to ethanol? Explain. 4. Fermentation to Produce Soy Sauce Soy sauce is prepared by fermenting a salted mixture of soybeans and wheat with several microorganisms, including yeast, over a period of 8 to 12 months. The resulting sauce (after solids are removed) is rich in lactate and ethanol. How are these two compounds produced? To prevent the soy sauce from having a strong vinegar taste (vinegar is dilute acetic acid), oxygen must be kept out of the fermentation tank. Why? 5. Equivalence of Triose Phosphates 14 C-Labeled glyceraldehyde 3-phosphate was added to a yeast extract. After a short time, fructose 1,6-bisphosphate labeled with 14 C at C-3 and C-4 was isolated. What was the location of the 14 C label in the starting glyceraldehyde 3-phosphate? Where did the second 14 C label in fructose 1,6-bisphosphate come from? Explain. 6. Glycolysis Shortcut Suppose you discovered a mu- tant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction: Would shortening the glycolytic pathway in this way benefit the cell? Explain. 7. Role of Lactate Dehydrogenase During strenuous ac- tivity, the demand for ATP in muscle tissue is vastly increased. In rabbit leg muscle or turkey flight muscle, the ATP is pro- duced almost exclusively by lactic acid fermentation. ATP is formed in the payoff phase of glycolysis by two reactions, pro- moted by phosphoglycerate kinase and pyruvate kinase. Sup- pose skeletal muscle were devoid of lactate dehydrogenase. Could it carry out strenuous physical activity; that is, could it generate ATP at a high rate by glycolysis? Explain. 8. Efficiency of ATP Production in Muscle The trans- formation of glucose to lactate in myocytes releases only about 7% of the free energy released when glucose is completely ox- idized to CO 2 and H 2 O. Does this mean that anaerobic glycol- ysis in muscle is a wasteful use of glucose? Explain. 9. Free-Energy Change for Triose Phosphate Oxidation The oxidation of glyceraldehyde 3-phosphate to 1,3-bisphos- phoglycerate, catalyzed by glyceraldehyde 3-phosphate dehy- drogenase, proceeds with an unfavorable equilibrium constant (KH11032 eq H11005 0.08; H9004GH11032H11034 H11005 6.3 kJ/mol), yet the flow through this point in the glycolytic pathway proceeds smoothly. How does the cell overcome the unfavorable equilibrium? 10. Arsenate Poisoning Arsenate is structurally and chemically similar to inorganic phosphate (P i ), and many en- zymes that require phosphate will also use arsenate. Organic compounds of arsenate are less stable than analogous phos- phate compounds, however. For example, acyl arsenates de- compose rapidly by hydrolysis: On the other hand, acyl phosphates, such as 1,3-bisphos- phoglycerate, are more stable and undergo further enzyme- catalyzed transformation in cells. (a) Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if phosphate were replaced by arsenate. (b) What would be the consequence to an organism if arsenate were substituted for phosphate? Arsenate is very toxic to most organisms. Explain why. 11. Requirement for Phosphate in Ethanol Fermenta- tion In 1906 Harden and Young, in a series of classic stud- ies on the fermentation of glucose to ethanol and CO 2 by extracts of brewer’s yeast, made the following observations. (1) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used. (2) During fermentation un- der these conditions, ethanol, CO 2 , and a hexose bisphosphate A B O H11001O H11002 OOO O B O O H11002 AsOCR H 2 O A B O H11001H11001 O H11002 OOO O B O O H11002H11001H11002 AsOCR HHO Glyceraldehyde 3-phosphate H11001 H 2 3-phosphoglycerate NAD H11001 NADH H11001 H H11001 Problems 8885d_c14_521-559 2/6/04 3:43 PM Page 557 mac76 mac76:385_reb: accumulated. (3) When arsenate was substituted for phos- phate, no hexose bisphosphate accumulated, but the fer- mentation proceeded until all the glucose was converted to ethanol and CO 2 . (a) Why did fermentation cease when the supply of phosphate was exhausted? (b) Why did ethanol and CO 2 accumulate? Was the con- version of pyruvate to ethanol and CO 2 essential? Why? Iden- tify the hexose bisphosphate that accumulated. Why did it accumulate? (c) Why did the substitution of arsenate for phosphate prevent the accumulation of the hexose bisphosphate yet al- low fermentation to ethanol and CO 2 to go to completion? (See Problem 10.) 12. Role of the Vitamin Niacin Adults engaged in stren- uous physical activity require an intake of about 160 g of car- bohydrate daily but only about 20 mg of niacin for optimal nutrition. Given the role of niacin in glycolysis, how do you explain the observation? 13. Metabolism of Glycerol Glycerol obtained from the breakdown of fat is metabolized by conversion to dihydroxy- acetone phosphate, a glycolytic intermediate, in two enzyme- catalyzed reactions. Propose a reaction sequence for glycerol metabolism. On which known enzyme-catalyzed reactions is your proposal based? Write the net equation for the conver- sion of glycerol to pyruvate according to your scheme. 14. Severity of Clinical Symptoms Due to Enzyme Deficiency The clinical symptoms of two forms of galactosemia—deficiency of galactokinase or of UDP-glucose:galactose 1-phosphate uridylyltransferase— show radically different severity. Although both types pro- duce gastric discomfort after milk ingestion, deficiency of the transferase also leads to liver, kidney, spleen, and brain dys- function and eventual death. What products accumulate in the blood and tissues with each type of enzyme deficiency? Estimate the relative toxicities of these products from the above information. 15. Muscle Wasting in Starvation One consequence of starvation is a reduction in muscle mass. What happens to the muscle proteins? 16. Pathway of Atoms in Gluconeogenesis A liver ex- tract capable of carrying out all the normal metabolic reac- tions of the liver is briefly incubated in separate experiments with the following 14 C-labeled precursors: Trace the pathway of each precursor through gluconeogene- sis. Indicate the location of 14 C in all intermediates and in the product, glucose. 17. Pathway of CO 2 in Gluconeogenesis In the first by- pass step of gluconeogenesis, the conversion of pyruvate to phosphoenolpyruvate, pyruvate is carboxylated by pyruvate carboxylase to oxaloacetate, which is subsequently decar- boxylated by PEP carboxykinase to yield phosphoenolpyru- vate. The observation that the addition of CO 2 is directly fol- lowed by the loss of CO 2 suggests that 14 C of 14 CO 2 would not be incorporated into PEP, glucose, or any intermediates in gluconeogenesis. However, when a rat liver preparation synthesizes glucose in the presence of 14 CO 2 , 14 C slowly ap- pears in PEP and eventually at C-3 and C-4 of glucose. How does the 14 C label get into PEP and glucose? (Hint: During gluconeogenesis in the presence of 14 CO 2 , several of the four- carbon citric acid cycle intermediates also become labeled.) 18. Energy Cost of a Cycle of Glycolysis and Gluco- neogenesis What is the cost (in ATP equivalents) of trans- forming glucose to pyruvate via glycolysis and back again to glucose via gluconeogenesis? 19. Glucogenic Substrates A common procedure for de- termining the effectiveness of compounds as precursors of glucose in mammals is to starve the animal until the liver glycogen stores are depleted and then administer the com- pound in question. A substrate that leads to a net increase in liver glycogen is termed glucogenic, because it must first be converted to glucose 6-phosphate. Show by means of known enzymatic reactions which of the following substances are glucogenic: 20. Ethanol Affects Blood Glucose Levels The consumption of alcohol (ethanol), especially after pe- riods of strenuous activity or after not eating for several hours, results in a deficiency of glucose in the blood, a con- dition known as hypoglycemia. The first step in the metabo- lism of ethanol by the liver is oxidation to acetaldehyde, cat- alyzed by liver alcohol dehydrogenase: CH 3 CH 2 OH H11001 NAD H11001 88n CH 3 CHO H11001 NADH H11001 H H11001 Explain how this reaction inhibits the transformation of lac- tate to pyruvate. Why does this lead to hypoglycemia? (a) Succinate, H5008 OOC CH 2 CH 2 (b) Glycerol, CH 2 OH C OH H CH 2 OH (c) Acetyl-CoA, CH 3 C S-CoA (d) Pyruvate, CH 3 C O O COO H5008 (e) Butyrate, CH 3 CH 2 CH 2 COO H5008 COO H5008 (a) [ 14 C]Bicarbonate, (b) [1- 14 C]Pyruvate, HO 14 C O H5008 O C O 14 COO H5008 CH 3 Glycerol HOCH 2 CH 2 OHO A A CO OH H Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway558 8885d_c14_558 2/9/04 7:05 AM Page 558 mac76 mac76:385_reb: Chapter 14 Problems 559 21. Blood Lactate Levels during Vigorous Exercise The concentrations of lactate in blood plasma before, during, and after a 400 m sprint are shown in the graph. (a) What causes the rapid rise in lactate concentration? (b) What causes the decline in lactate concentration af- ter completion of the sprint? Why does the decline occur more slowly than the increase? (c) Why is the concentration of lactate not zero during the resting state? 22. Relationship between Fructose 1,6-Bisphosphatase and Blood Lactate Levels A congenital defect in the liver enzyme fructose 1,6-bisphosphatase results in abnormally high levels of lactate in the blood plasma. Explain. Blood [lactate] ( M ) 0 150 Time (min) 100 50 6040 Before 0 200 Run After 20 H9262 23. Effect of Phloridzin on Carbohydrate Metabolism Phloridzin, a toxic glycoside from the bark of the pear tree, blocks the normal reabsorption of glucose from the kidney tubule, thus causing blood glucose to be almost completely excreted in the urine. In an experiment, rats fed phloridzin and sodium succinate excreted about 0.5 mol of glucose (made by gluconeogenesis) for every 1 mol of sodium succi- nate ingested. How is the succinate transformed to glucose? Explain the stoichiometry. 24. Excess O 2 Uptake during Gluconeogenesis Lactate absorbed by the liver is converted to glucose, with the input of 6 mol of ATP for every mole of glucose produced. The ex- tent of this process in a rat liver preparation can be moni- tored by administering [ 14 C]lactate and measuring the amount of [ 14 C]glucose produced. Because the stoichiometry of O 2 consumption and ATP production is known (about 5 ATP per O 2 ), we can predict the extra O 2 consumption above the nor- mal rate when a given amount of lactate is administered. How- ever, when the extra O 2 used in the synthesis of glucose from lactate is actually measured, it is always higher than predicted by known stoichiometric relationships. Suggest a possible ex- planation for this observation. O C O OH OH OH HO HOH H H OHH HOCH 2 CH 2 H O Phloridzin CH 2 8885d_c14_521-559 2/6/04 3:43 PM Page 559 mac76 mac76:385_reb: chapter M etabolic regulation, a central theme in biochem- istry, is one of the most remarkable features of a living cell. Of the thousands of enzyme-catalyzed reac- tions that can take place in a cell, there is probably not one that escapes some form of regulation. Although it is convenient (and perhaps essential) in writing a text- book to divide metabolic processes into “pathways” that play discrete roles in the cell’s economy, no such sepa- ration exists inside the cell. Rather, each of the path- ways we discuss in this book is inextricably intertwined with all the other cellular pathways in a multidimen- sional network of reactions (Fig. 15–1). For example, in Chapter 14 we discussed three possible fates for glu- cose 6-phosphate in a hepatocyte: passage into glycol- ysis for the production of ATP, passage into the pentose phosphate pathway for the production of NADPH and pentose phosphates, or hydrolysis to glucose and phos- phate to replenish blood glucose. In fact, glucose 6-phos- phate has a number of other possible fates; it may, for example, be used to synthesize other sugars, such as glucosamine, galactose, galactosamine, fucose, and neu- raminic acid, for use in protein glycosylation, or it may be partially degraded to provide acetyl-CoA for fatty acid and sterol synthesis. In the extreme case, the bac- terium Escherichia coli can use glucose to produce the carbon skeleton of every one of its molecules. When a cell “decides” to use glucose 6-phosphate for one pur- pose, that decision affects all the other pathways for which glucose 6-phosphate is a precursor or intermedi- ate; any change in the allocation of glucose 6-phosphate to one pathway affects, directly or indirectly, the metabolite flow through all the others. Such changes in allocation are common in the life of a cell. Louis Pasteur was the first to describe the large (greater than tenfold) increase in glucose consumption by a yeast culture when it was shifted from aerobic to anaerobic conditions. This phenomenon, called the PRINCIPLES OF METABOLIC REGULATION: GLUCOSE AND GLYCOGEN 15.1 The Metabolism of Glycogen in Animals 562 15.2 Regulation of Metabolic Pathways 571 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 575 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown 583 15.5 Analysis of Metabolic Control 591 Formation of liver glycogen from lactic acid is thus seen to establish an important connection between the metabolism of the muscle and that of the liver. Muscle glycogen becomes available as blood sugar through the intervention of the liver, and blood sugar in turn is converted into muscle glycogen. There exists therefore a complete cycle of the glucose molecule in the body . . . Epinephrine was found to accelerate this cycle in the direction of muscle glycogen to liver glycogen . . . Insulin, on the other hand, was found to accelerate the cycle in the direction of blood glucose to muscle glycogen. —C. F. Cori and G. T. Cori, article in Journal of Biological Chemistry, 1929 560 15 8885d_c15_560 2/26/04 1:59 PM Page 560 mac76 mac76:385_reb: Pasteur effect, occurs without a significant change in the concentration of ATP or any of the hundreds of meta- bolic intermediates and products derived from glucose. A similar change takes place in cells of skeletal muscle when a sprinter leaves the starting blocks. The ability of a cell to carry out all these interlocking metabolic processes simultaneously—obtaining every product in the amount needed and at the right time, in the face of major perturbations from outside, and without generat- ing leftovers—is an astounding accomplishment. In this chapter we look at mechanisms of metabolic regulation, using the pathways in which glucose is an intermediate to illustrate some general principles. First we consider the pathways by which glycogen is synthe- sized and broken down, a very well-studied case of meta- bolic regulation. Then we look at the general roles of regulation in achieving metabolic homeostasis. Focus- ing on the pathways that connect pyruvate with glyco- gen in both directions, we next consider the specific reg- ulatory properties of the participating enzymes and the ways in which the cell accomplishes coordinated regu- lation of catabolic and anabolic pathways. Finally, we discuss metabolic control analysis, a system for treating complex metabolic interactions quantitatively, and con- sider some surprising results of its application. In selecting carbohydrate metabolism to illustrate the principles of metabolic regulation, we have artifi- cially separated the metabolism of fats and carbohy- drates. In fact, these two activities are very tightly in- tegrated, as we shall see in Chapter 23. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen 561 Metabolism of Other Amino Acids Amino Acid Metabolism Lipid Metabolism Carbohydrate Metabolism Energy Metabolism Metabolism of Complex Carbohydrates Metabolism of Complex Lipids Metabolism of Cofactors and Vitamins Nucleotide Metabolism METABOLIC PATHWAYS Biosynthesis of Secondary Metabolites Biodegradation of Xenobiotics FIGURE 15–1 Metabolism as a three- dimensional meshwork. A typical eukaryotic cell has the capacity to make about 30,000 different proteins, which catalyze thousands of different reactions involving many hundreds of metabolites, most shared by more than one “pathway.” This overview image of metabolic pathways is from the online KEGG (Kyoto Encyclopedia of Genes and Genomes) PATHWAY database (www.genome.ad.jp/kegg/pathway/map /map01100.html). Each area can be further expanded for increasingly detailed information, to the level of specific enzymes and intermediates. 8885d_c15_560-600 2/26/04 9:03 AM Page 561 mac76 mac76:385_reb: 15.1 The Metabolism of Glycogen in Animals In a wide range of organisms, excess glucose is con- verted to polymeric forms for storage—glycogen in ver- tebrates and many microorganisms, starch in plants. In vertebrates, glycogen is found primarily in the liver and skeletal muscle; it may represent up to 10% of the weight of liver and 1% to 2% of the weight of muscle. If this much glucose were dissolved in the cytosol of a hepatocyte, its concentration would be about 0.4 M, enough to dominate the osmotic properties of the cell. When stored as a long polymer (glycogen), however, the same mass of glucose has a concentration of only 0.01 H9262M. Glycogen is stored in large cytosolic granules. The elementary particle of glycogen, the H9252-particle, about 21 nm in diameter, consists of up to 55,000 glucose residues with about 2,000 nonreducing ends. Twenty to 40 of these particles cluster together to form H9251-rosettes, easily seen with the microscope in tissue samples from well-fed animals (Fig. 15–2) but essentially absent after a 24-hour fast. The glycogen in muscle is there to provide a quick source of energy for either aerobic or anaerobic metab- olism. Muscle glycogen can be exhausted in less than an hour during vigorous activity. Liver glycogen serves as a reservoir of glucose for other tissues when dietary glu- cose is not available (between meals or during a fast); this is especially important for the neurons of the brain, which cannot use fatty acids as fuel. Liver glycogen can be depleted in 12 to 24 hours. In humans, the total amount of energy stored as glycogen is far less than the amount stored as fat (triacylglycerol) (see Table 23–5), but fats cannot be converted to glucose in mammals and cannot be catabolized anaerobically. Glycogen granules are complex aggregates of glyco- gen and the enzymes that synthesize it and degrade it, as well as the machinery for regulating these enzymes. The general mechanisms for storing and mobilizing glycogen are the same in muscle and liver, but the en- zymes differ in subtle yet important ways that reflect the different roles of glycogen in the two tissues. Glyco- gen is also obtained in the diet and broken down in the gut, and this involves a separate set of hydrolytic enzymes that convert glycogen (and starch) to free glucose. The transformations of glucose discussed in this chapter and in Chapter 14 are central to the metabo- lism of most organisms, microbial, animal, or plant. We begin with a discussion of the catabolic pathways from glycogen to glucose 6-phosphate (glycogenolysis) and from glucose 6-phosphate to pyruvate (glycolysis), then turn to the anabolic pathways from pyruvate to glucose (gluconeogenesis) and from glucose to glyco- gen (glycogenesis). Glycogen Breakdown Is Catalyzed by Glycogen Phosphorylase In skeletal muscle and liver, the glucose units of the outer branches of glycogen enter the glycolytic pathway through the action of three enzymes: glycogen phos- phorylase, glycogen debranching enzyme, and phos- phoglucomutase. Glycogen phosphorylase catalyzes the reaction in which an (H92511n4) glycosidic linkage between two glucose residues at a nonreducing end of glycogen undergoes attack by inorganic phosphate (P i ), remov- ing the terminal glucose residue as H9251-D-glucose 1-phos- phate (Fig. 15–3). This phosphorolysis reaction is different from the hydrolysis of glycosidic bonds by amylase during intestinal degradation of dietary glyco- gen and starch. In phosphorolysis, some of the energy of the glycosidic bond is preserved in the formation of the phosphate ester, glucose 1-phosphate. Pyridoxal phosphate is an essential cofactor in the glycogen phosphorylase reaction; its phosphate group acts as a general acid catalyst, promoting attack by P i on the glycosidic bond. (This is an unusual role for this cofactor; its more typical role is as a cofactor in amino acid metabolism; see Fig. 18–6.) Glycogen phosphorylase acts repetitively on the nonreducing ends of glycogen branches until it reaches a point four glucose residues away from an (H92511n6) branch point (see Fig. 7–15), where its action stops. Further degradation by glycogen phosphorylase can oc- cur only after the debranching enzyme, formally known as oligo (H92511n6) to (H92511n4) glucantrans- ferase, catalyzes two successive reactions that transfer Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen562 FIGURE 15–2 Glycogen granules in a hepatocyte. Glycogen is a stor- age form of carbohydrate in cells, especially hepatocytes, as illustrated here. Glycogen appears as electron-dense particles, often in aggre- gates or rosettes. In hepatocytes the glycogen is closely associated with tubules of the smooth endoplasmic reticulum. Many mitochondria are also present. 8885d_c15_562 2/26/04 2:52 PM Page 562 mac76 mac76:385_reb: branches (Fig. 15–4). Once these branches are trans- ferred and the glucosyl residue at C-6 is hydrolyzed, glycogen phosphorylase activity can continue. Glucose 1-Phosphate Can Enter Glycolysis or, in Liver, Replenish Blood Glucose Glucose 1-phosphate, the end product of the glycogen phosphorylase reaction, is converted to glucose 6-phos- phate by phosphoglucomutase, which catalyzes the reversible reaction Glucose 1-phosphate glucose 6-phosphate Initially phosphorylated at a Ser residue, the enzyme do- nates a phosphoryl group to C-6 of the substrate, then accepts a phosphoryl group from C-1 (Fig. 15–5). The glucose 6-phosphate formed from glycogen in skeletal muscle can enter glycolysis and serve as an en- ergy source to support muscle contraction. In liver, z y 15.1 The Metabolism of Glycogen in Animals 563 H11001 O Glycogen shortened by one residue (glucose) nH110021 Glycogen chain (glucose) n A O B OPO H11002 O H11002 Glucose 1-phosphate 32 41 6 O Nonreducing end 5 OO HO H H H H OH H CH 2 OH CH 2 OH CH 2 OH OH O H H H H OH H OH O Nonreducing end OO HO H H H H OH H CH 2 OH CH 2 OH OH O H H H H OH H OH O O H H H H OH H OH 32 41 6 O 5 O HO H H H H OH H CH 2 OH OH glycogen phosphorylase P i O O Glucose 1-phosphate molecules (α1→ 6) glucosidase activity of debranching enzyme transferase activity of debranching enzyme Unbranched (α1→ 4) polymer; substrate for further phosphorylase action glycogen phosphorylase (α1→ 6) linkage Nonreducing ends Glycogen Glucose FIGURE 15–3 Removal of a terminal glucose residue from the nonreducing end of a glycogen chain by glycogen phosphorylase. This process is repeti- tive; the enzyme removes successive glucose residues until it reaches the fourth glucose unit from a branch point (see Fig. 15–4). FIGURE 15–4 Glycogen breakdown near an (H92511n6) branch point. Following sequential removal of terminal glucose residues by glyco- gen phosphorylase (see Fig. 15–3), glucose residues near a branch are removed in a two-step process that requires a bifunctional “de- branching enzyme.” First, the transferase activity of the enzyme shifts a block of three glucose residues from the branch to a nearby nonre- ducing end, to which they are reattached in (H92511n4) linkage. The sin- gle glucose residue remaining at the branch point, in (H92511n6) linkage, is then released as free glucose by the enzyme’s (H92511n6) glucosidase activity. The glucose residues are shown in shorthand form, which omits the OH, OOH, and OCH 2 OH groups from the pyranose rings. 8885d_c15_560-600 2/26/04 9:03 AM Page 563 mac76 mac76:385_reb: glycogen breakdown serves a different purpose: to re- lease glucose into the blood when the blood glucose level drops, as it does between meals. This requires an enzyme, glucose 6-phosphatase, that is present in liver and kidney but not in other tissues. The enzyme is an integral membrane protein of the endoplasmic reticu- lum, predicted to contain nine transmembrane helices, with its active site on the lumenal side of the ER. Glu- cose 6-phosphate formed in the cytosol is transported into the ER lumen by a specific transporter (T1) (Fig. 15–6) and hydrolyzed at the lumenal surface by the glu- cose 6-phosphatase. The resulting P i and glucose are thought to be carried back into the cytosol by two dif- ferent transporters (T2 and T3), and the glucose leaves Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen564 O O – O O – P Glucose 1-phosphate Glucose 1,6-bisphosphate Glucose 6-phosphate 2 1 Phosphoglucomutase HOCH 2 HH HO OH OH H H HOH O O O – O O – P O O – O O – P CH 2 HH HO OH H H HOH O O O – O – O P O O – O O – P CH 2 HH HO OH H H HOH O OHSer Ser FIGURE 15–5 Reaction catalyzed by phosphogluco- mutase. The reaction begins with the enzyme phosphorylated on a Ser residue. In step 1 , the enzyme donates its phosphoryl group (green) to glucose 1-phosphate, producing glucose 1,6-bisphos- phate. In step 2 , the phosphoryl group at C-1 of glucose 1,6-bisphosphate (red) is transferred back to the enzyme, re-forming the phosphoenzyme and producing glucose 6-phosphate. FIGURE 15–6 Hydrolysis of glucose 6-phosphate by glucose 6- phosphatase of the ER. The catalytic site of glucose 6-phosphatase faces the lumen of the ER. A glucose 6-phosphate (G6P) transporter (T1) carries the substrate from the cytosol to the lumen, and the prod- ucts glucose and P i pass to the cytosol on specific transporters (T2 and T3). Glucose leaves the cell via the GLUT2 transporter in the plasma membrane. Cytosol ER lumen G6P G6P G6P transporter (T1) Glucose 6-phosphatase Glucose transporter (T2) Glucose Glucose P i P i Plasma membrane GLUT2 Capillary Increased blood glucose concentration P i transporter (T3) 8885d_c15_560-600 2/26/04 9:03 AM Page 564 mac76 mac76:385_reb: the hepatocyte via yet another transporter in the plasma membrane (GLUT2). Notice that by having the active site of glucose 6-phosphatase inside the ER lumen, the cell separates this reaction from the process of glycol- ysis, which takes place in the cytosol and would be aborted by the action of glucose 6-phosphatase. Genetic defects in either glucose 6-phosphatase or T1 lead to serious derangement of glycogen metabolism, resulting in type Ia glycogen storage disease (Box 15–1). Because muscle and adipose tissue lack glucose 6-phosphatase, they cannot convert the glucose 6- phosphate formed by glycogen breakdown to glucose, and these tissues therefore do not contribute glucose to the blood. The Sugar Nucleotide UDP-Glucose Donates Glucose for Glycogen Synthesis Many of the reactions in which hexoses are transformed or polymerized involve sugar nucleotides, compounds in which the anomeric carbon of a sugar is activated by attachment to a nucleotide through a phosphate ester linkage. Sugar nucleotides are the substrates for poly- merization of monosaccharides into disaccharides, glycogen, starch, cellulose, and more complex extracel- lular polysaccharides. They are also key intermediates in the production of the aminohexoses and deoxyhex- oses found in some of these polysaccharides, and in the synthesis of vitamin C (L-ascorbic acid). The role of sugar nucleotides in the biosynthesis of glycogen and many other carbohydrate derivatives was first discov- ered by the Argentine biochemist Luis Leloir. The suitability of sugar nucleotides for biosynthetic reactions stems from several properties: 1. Their formation is metabolically irreversible, con- tributing to the irreversibility of the synthetic pathways in which they are intermediates. The condensation of a nucleoside triphosphate with a hexose 1-phosphate to form a sugar nucleotide has a small positive free-energy change, but the reaction releases PP i , which is rapidly hydrolyzed by inorganic pyrophosphatase in a reaction that is strongly exergonic (H9004GH11032H11034 H11005 H1100219.2 kJ/mol; Fig. 15–7). This keeps the cellular concentration of PP i low, ensuring that the actual free-energy change in 15.1 The Metabolism of Glycogen in Animals 565 D- O HN OH H H HO H CH 2 OH H5008 OPO O H P O O H5008 CH 2 H H OH H NOO OH HH O HO O O UDP-glucose (a sugar nucleotide) Glucosyl group Uridine FIGURE 15–7 Formation of a sugar nucleotide. A condensation reaction occurs between a nucleoside triphosphate (NTP) and a sugar phosphate. The negatively charged oxygen on the sugar phosphate serves as a nucleophile, attacking the H9251 phosphate of the nucleoside triphosphate and displacing pyrophosphate. The reaction is pulled in the forward direction by the hydrolysis of PP i by inorganic pyrophosphatase. Sugar O P H11002 O H11001 Ribose Base Sugar phosphate NDP-sugar inorganic Sugar nucleotide (NDP-sugar) NTP Phosphate (P i ) Pyrophosphate (PP i ) O H11002 OH PO P O H11002 O OP O O H11002 O O H11002 OP H11002 O O PO O H11002 O O H11002 O H11002 Sugar O O H11002 P O H11002 O O Ribose Base O H11002 OP O P H11002 O O O H11002 O Net reaction: Sugar phosphate H11001 NTP NDP-sugar H11001 2P i pyrophosphorylase pyrophosphatase 2 Luis Leloir, 1906–1987 8885d_c15_560-600 2/26/04 9:03 AM Page 565 mac76 mac76:385_reb: the cell is favorable. In effect, rapid removal of the product, driven by the large, negative free-energy change of PP i hydrolysis, pulls the synthetic reac- tion forward, a common strategy in biological polymerization reactions. 2. Although the chemical transformations of sugar nucleotides do not involve the atoms of the nu- cleotide itself, the nucleotide moiety has many groups that can undergo noncovalent interactions with enzymes; the additional free energy of bind- ing can contribute significantly to catalytic activity (Chapter 6; see also p. 301). 3. Like phosphate, the nucleotidyl group (UMP or AMP, for example) is an excellent leaving group, facilitating nucleophilic attack by activating the sugar carbon to which it is attached. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen566 BOX 15–1 WORKING IN BIOCHEMISTRY Carl and Gerty Cori: Pioneers in Glycogen Metabolism and Disease Much of what is written in present-day biochemistry textbooks about the metabolism of glycogen was dis- covered between about 1925 and 1950 by the re- markable husband and wife team of Carl F. Cori and Gerty T. Cori. Both trained in medicine in Europe at the end of World War I (she completed premedical studies and medical school in one year!). They left Europe together in 1922 to establish research labora- tories in the United States, first for nine years in Buffalo, New York, at what is now the Roswell Park Memorial Institute, then from 1931 until the end of their lives at Washington University in St. Louis. In their early physiological studies of the origin and fate of glycogen in animal muscle, the Coris demonstrated the conversion of glycogen to lactate in tissues, movement of lactate in the blood to the liver, and, in the liver, reconversion of lactate to glycogen— a pathway that came to be known as the Cori cycle (see Fig. 23–18). Pursuing these observations at the biochemical level, they showed that glycogen was mo- bilized in a phosphorolysis reaction catalyzed by the enzyme they discovered, glycogen phosphorylase. They identified the product of this reaction (the “Cori ester”) as glucose 1-phosphate and showed that it could be reincorporated into glycogen in the reverse reaction. Although this did not prove to be the reac- tion by which glycogen is synthesized in cells, it was the first in vitro demonstration of the synthesis of a macromolecule from simple monomeric subunits, and it inspired others to search for polymerizing enzymes. Arthur Kornberg, discoverer of the first DNA poly- merase, has said of his experience in the Coris’ lab, “Glycogen phosphorylase, not base pairing, was what led me to DNA polymerase.” Gerty Cori became interested in human genetic diseases in which too much glycogen is stored in the liver. She was able to identify the biochemical defect in several of these diseases and to show that these diseases could be diagnosed by assays of the en- zymes of glycogen metabolism in small samples of tis- sue obtained by biopsy. Table 1 summarizes what we now know about 13 genetic diseases of this sort. ■ Carl and Gerty Cori shared the Nobel Prize in Physiology or Medicine in 1947 with Bernardo Hous- say of Argentina, who was cited for his studies of hor- monal regulation of carbohydrate metabolism. The Cori laboratories in St. Louis became an international center of biochemical research in the 1940s and 1950s, and at least six scientists who trained with the Coris became Nobel laureates: Arthur Kornberg (for DNA synthesis, 1959), Severo Ochoa (for RNA synthesis, 1959), Luis Leloir (for the role of sugar nucleotides inThe Coris in Gerty Cori’s laboratory, around 1947. 8885d_c15_560-600 2/26/04 9:04 AM Page 566 mac76 mac76:385_reb: 4. By “tagging” some hexoses with nucleotidyl groups, cells can set them aside in a pool for one purpose (glycogen synthesis, for example), sepa- rate from hexose phosphates destined for another purpose (such as glycolysis). Glycogen synthesis takes place in virtually all animal tissues but is especially prominent in the liver and skele- tal muscles. The starting point for synthesis of glycogen is glucose 6-phosphate. As we saw in Chapter 14, this can be derived from free glucose in a reaction catalyzed by the isozymes hexokinase I and hexokinase II in muscle and hexokinase IV (glucokinase) in liver: D-Glucose H11001 ATP 88n D-glucose 6-phosphate H11001 ADP However, some ingested glucose takes a more roundabout path to glycogen. It is first taken up by erythrocytes and converted to lactate glycolytically; the lactate is then 15.1 The Metabolism of Glycogen in Animals 567 polysaccharide synthesis, 1970), Earl Sutherland (for the discovery of cAMP in the regulation of carbohy- drate metabolism, 1971), Christian de Duve (for sub- cellular fractionation, 1974), and Edwin Krebs (for the discovery of phosphorylase kinase, 1991). Primary organ Type (name) Enzyme affected affected Symptoms Type 0 Glycogen synthase Liver Low blood glucose, high ketone bodies, early death Type Ia (von Gierke’s) Glucose 6-phosphatase Liver Enlarged liver, kidney failure Type Ib Microsomal glucose Liver As in Ia; also high 6-phosphate translocase susceptibility to bacterial infections Type Ic Microsomal P i Liver As in Ia transporter Type II (Pompe’s) Lysosomal glucosidase Skeletal and Infantile form: death by age 2; cardiac muscle juvenile form: muscle defects (myopathy); adult form: as in muscular dystrophy Type IIIa (Cori’s or Forbes’s) Debranching enzyme Liver, skeletal Enlarged liver in infants; and cardiac myopathy muscle Type IIIb Liver debranching Liver Enlarged liver in infants enzyme (muscle enzyme normal) Type IV (Andersen’s) Branching enzyme Liver, skeletal Enlarged liver and spleen, muscle myoglobin in urine Type V (McArdle’s) Muscle phosphorylase Skeletal muscle Exercise-induced cramps and pain; myoglobin in urine Type VI (Hers’s) Liver phosphorylase Liver Enlarged liver Type VII (Tarui’s) Muscle PFK-1 Muscle, As in V; also hemolytic erythrocytes anemia Type VIb, VIII, or IX Phosphorylase kinase Liver, leukocytes, Enlarged liver muscle Type XI (Fanconi-Bickel) Glucose transporter Liver Failure to thrive, enlarged (GLUT2) liver, rickets, kidney dysfunction TABLE 1 Glycogen Storage Diseases of Humans 8885d_c15_560-600 2/26/04 9:04 AM Page 567 mac76 mac76:385_reb: taken up by the liver and converted to glucose 6-phos- phate by gluconeogenesis. To initiate glycogen synthesis, the glucose 6- phosphate is converted to glucose 1-phosphate in the phosphoglucomutase reaction: Glucose 6-phosphate glucose 1-phosphate The product of this reaction is converted to UDP- glucose by the action of UDP-glucose pyrophosphor- ylase, in a key step of glycogen biosynthesis: Glucose 1-phosphate H11001 UTP 88n UDP-glucose H11001 PP i Notice that this enzyme is named for the reverse reaction; in the cell, the reaction proceeds in the direction of UDP- glucose formation, because pyrophosphate is rapidly hydrolyzed by inorganic pyrophosphatase (Fig. 15–7). UDP-glucose is the immediate donor of glucose res- idues in the reaction catalyzed by glycogen synthase, which promotes the transfer of the glucose residue from UDP-glucose to a nonreducing end of a branched glyco- z y gen molecule (Fig. 15–8). The overall equilibrium of the path from glucose 6-phosphate to lengthened glycogen greatly favors synthesis of glycogen. Glycogen synthase cannot make the (H92511n6) bonds found at the branch points of glycogen; these are formed by the glycogen-branching enzyme, also called amylo (1n4) to (1n6) transglycosylase or glycosyl- (4n6)-transferase. The glycogen-branching enzyme catalyzes transfer of a terminal fragment of 6 or 7 glu- cose residues from the nonreducing end of a glycogen branch having at least 11 residues to the C-6 hydroxyl group of a glucose residue at a more interior position of the same or another glycogen chain, thus creating a new branch (Fig. 15–9). Further glucose residues may be added to the new branch by glycogen synthase. The biological effect of branching is to make the glycogen molecule more soluble and to increase the number of nonreducing ends. This increases the number of sites accessible to glycogen phosphorylase and glycogen syn- thase, both of which act only at nonreducing ends. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen568 OHO 3 5 6 41 2 H OH HO H H CH 2 OH H O H O H11002 O P O P O O O H11002 UDP-glucose glycogen H 41 H OH OH H H CH 2 OH O H O CH 2 Uracil O H OHOH H H CH 2 OH O H 1 HOH H H 4 HO HOH OO H 41 H OH OH H H CH 2 OH O H O CH 2 OH O H 1 HOH H H 4 HO HOH O UDP CH 2 OH O HOH H H 4 HOH New nonreducing Elongated glycogen 1 synthase end with n H11001 1 residues H H Nonreducing end of a glycogen chain with n residues (n H11022 4) FIGURE 15–8 Glycogen synthesis. A glycogen chain is elongated by glycogen synthase. The en- zyme transfers the glucose residue of UDP-glucose to the nonreducing end of a glycogen branch (see Fig. 7–15) to make a new (H92511n4) linkage. 8885d_c15_560-600 2/26/04 9:04 AM Page 568 mac76 mac76:385_reb: 15.1 The Metabolism of Glycogen in Animals 569 O O O O O O O O O O O O O O O O O O O OO O O O O O O O O O O O O O O OO O O O O O HO HO HO glycogen-branching Glycogen core (1 4) Glycogen core Nonreducing end ( 1 6) Branch point enzyme Nonreducing end Nonreducing end H9251 H9251 FIGURE 15–9 Branch synthesis in glycogen. The glycogen-branching enzyme (also called amylo (1n4) to (1n6) transglycosylase or glycosyl-(4n6)-transferase) forms a new branch point during glycogen synthesis. FIGURE 15–10 Glycogenin structure. (PDB 1D 1772) Muscle glycogenin (M r 37,000) forms dimers in solution. Humans have a second isoform in liver, glycogenin-2. The substrate, UDP-glucose (shown as a red ball-and-stick structure), is bound to a Rossman fold near the amino terminus and is some distance from the Tyr 194 residues (turquoise)—15 ? from that in the same monomer, 12 ? from that in the dimeric partner. Each UDP-glucose is bound through its phosphates to a Mn 2H11001 ion (green) that is essential to catalysis. Mn 2H11001 is believed to function as an electron-pair acceptor (Lewis acid) to stabilize the leaving group, UDP. The glycosidic bond in the product has the same configuration about the C-1 of glucose as the substrate UDP-glucose, suggesting that the transfer of glucose from UDP to Tyr 194 occurs in two steps. The first step is probably a nucleophilic attack by Asp 162 (orange), forming a temporary intermediate with inverted configuration. A second nucleophilic attack by Tyr 194 then restores the starting configuration. cule is the transfer of a glucose residue from UDP- glucose to the hydroxyl group of Tyr 194 of glycogenin, catalyzed by the protein’s intrinsic glucosyltransferase activity (Fig. 15–11a). The nascent chain is extended by the sequential addition of seven more glucose residues, each derived from UDP-glucose; the reactions are cat- alyzed by the chain-extending activity of glycogenin. At this point, glycogen synthase takes over, further ex- tending the glycogen chain. Glycogenin remains buried within the particle, covalently attached to the single re- ducing end of the glycogen molecule (Fig. 15–11b). Glycogenin Primes the Initial Sugar Residues in Glycogen Glycogen synthase cannot initiate a new glycogen chain de novo. It requires a primer, usually a preformed (H92511n4) polyglucose chain or branch having at least eight glucose residues. How is a new glycogen molecule initiated? The intriguing protein glycogenin (Fig. 15–10) is both the primer on which new chains are as- sembled and the enzyme that catalyzes their assembly. The first step in the synthesis of a new glycogen mole- 8885d_c15_560-600 2/26/04 9:04 AM Page 569 mac76 mac76:385_reb: SUMMARY 15.1 The Metabolism of Glycogen in Animals ■ Glycogen is stored in muscle and liver as large particles. Contained within the particles are the enzymes that metabolize glycogen, as well as regulatory enzymes. ■ Glycogen phosphorylase catalyzes phosphorolytic cleavage at the nonreducing ends of glycogen chains, producing glucose 1-phosphate. The debranching enzyme transfers branches onto main chains and releases the residue at the (H92511n6) branch as free glucose. ■ Phosphoglucomutase interconverts glucose 1-phosphate and glucose 6-phosphate. Glucose 6-phosphate can enter glycolysis or, in liver, can be converted to free glucose by glucose 6-phosphatase in the endoplasmic reticulum, then released to replenish blood glucose. ■ The sugar nucleotide UDP-glucose donates glucose residues to the nonreducing end of glycogen in the reaction catalyzed by glycogen synthase. A separate branching enzyme produces the (H92511n6) linkages at branch points. ■ New glycogen particles begin with the auto- catalytic formation of a glycosidic bond between the glucose of UDP-glucose and a Tyr residue in the protein glycogenin, followed by addition of several glucose residues to form a primer that can be acted upon by glycogen synthase. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen570 O O O P CH 2 OH HH HO OH H H HOH O O O –– O O P O O O P CH 2 OH HH HO OH H H HOH O O O –– O O P CH 2 OH H HO OH H H HOH O O (a) HO : Tyr 194 Glycogenin Ribose Uracil UDP-glucose UDP-glucose UDP glucosyltransferase activity chain-extending activity Repeats six times : Ribose Uracil UDP-glucose UDP (b) Each chain has 12 to 14 glucose residues glycogenin primer second tier fourth tier third tier outer tier (unbranched) G G MECHANISM FIGURE 15–11 Glycogenin and the structure of the glycogen particle. (a) Glycogenin catalyzes two distinct reactions. Initial attack by the hydroxyl group of Tyr 194 on C-1 of the glucosyl moiety of UDP-glucose results in a glucosylated Tyr residue. The C-1 of another UDP-glucose molecule is now attacked by the C-4 hydroxyl group of the terminal glucose, and this sequence repeats to form a nascent glycogen molecule of eight glucose residues attached by (H92511n4) glycosidic linkages. (b) Structure of the glycogen particle. Starting at a central glycogenin molecule, glycogen chains (12 to 14 residues) extend in tiers. Inner chains have two (H92511n6) branches each. Chains in the outer tier are unbranched. There are 12 tiers in a mature glycogen particle (only 5 are shown here), consisting of about 55,000 glucose residues in a molecule of about 21 nm diameter and M r 10 7 . 8885d_c15_560-600 2/26/04 9:04 AM Page 570 mac76 mac76:385_reb: 15.2 Regulation of Metabolic Pathways The pathways of glycogen metabolism provide, in the catabolic direction, the energy essential to oppose the forces of entropy and, in the anabolic direction, biosyn- thetic precursors and a storage form of metabolic en- ergy. These reactions are so important to survival that very complex regulatory mechanisms have evolved to ensure that metabolites move through each pathway in the correct direction and at the correct rate to exactly match the cell’s or the organism’s current circum- stances, and that appropriate adjustments are made in the rate of metabolite flow through the whole pathway if external circumstances change. Circumstances do change, sometimes dramatically. The demand for ATP production in muscle may increase 100-fold in a few seconds in response to exercise. The availability of oxygen may decrease due to hypoxia (diminished delivery of oxygen to tissues) or ischemia (diminished flow of blood to tissues). The relative pro- portions of carbohydrate, fat, and protein in the diet vary from meal to meal, and the supply of fuels obtained in the diet is intermittent, requiring metabolic adjust- ments between meals and during starvation. Wound healing requires huge amounts of energy and biosyn- thetic precursors. Living Cells Maintain a Dynamic Steady State Fuels such as glucose enter a cell, and waste products such as CO 2 leave, but the mass and the gross compo- sition of a typical cell do not change appreciably over time; cells and organisms exist in a dynamic steady state, but not at equilibrium with their surroundings. At the molecular level, this means that for each metabolic reaction in a pathway, the substrate is provided by the preceding reaction at the same rate at which it is con- verted to product. Thus, although the rate of metabo- lite flow, or flux, through this step of the pathway may be high, the concentration of substrate, S, remains con- stant. For the reaction v 1 v 2 A 88n S 88n P when v 1 H11005 v 2 , [S] is constant. When the steady state is disturbed by some change in external circumstances or energy supply, the tem- porarily altered fluxes through individual metabolic pathways trigger regulatory mechanisms intrinsic to each pathway. The net effect of all these adjustments is to return the organism to a new steady state—to achieve homeostasis. Regulatory Mechanisms Evolved under Strong Selective Pressures In the course of evolution, organisms have acquired a remarkable collection of regulatory mechanisms for maintaining homeostasis at the molecular, cellular, and organismal level. The importance of metabolic regula- tion to an organism is reflected in the relative propor- tion of genes that encode regulatory machinery—in hu- mans, about 4,000 genes (~12% of all genes) encode regulatory proteins, including a variety of receptors, regulators of gene expression, and about 500 different protein kinases! These regulatory mechanisms act over different time scales (from seconds to days) and have different sensitivities to external changes. In many cases, the mechanisms overlap: one enzyme is subject to regulation by several different mechanisms. After the protection of its DNA from damage, per- haps nothing is more important to a cell than maintain- ing a constant supply and concentration of ATP. Many ATP-using enzymes have K m values between 0.1 and 1mM, and the ATP concentration in a typical cell is about 5 mM. If [ATP] were to drop significantly, the rates of hundreds of reactions that involve ATP would decrease, and the cell would probably not survive. Furthermore, because ATP is converted to ADP or AMP when “spent” to accomplish cellular work, the [ATP]/[ADP] ratio pro- foundly affects all reactions that employ these cofactors. The same is true for other important cofactors, such as NADH/NAD H11001 and NADPH/NADP H11001 . For example, con- sider the reaction catalyzed by hexokinase: ATP H11001 glucose 88n ADP H11001 glucose 6-phosphate KH11032 eq H11005H110052 H11003 10 3 Note that this expression is true only when reactants and products are at their equilibrium concentrations, where H9004GH11032H110050. At any other set of concentrations, H9004GH11032 is not zero. Recall (from Chapter 13) that the ratio of products to substrates (the mass action ratio, Q) de- termines the magnitude and sign of H9004GH11032 and therefore the amount of free energy released during the reaction: H9004GH11032H11005H9004GH11032H11034 H11001 RT ln Because an alteration of this mass action ratio pro- foundly influences every reaction that involves ATP, or- ganisms have been under strong evolutionary pressure to develop regulatory mechanisms that respond to the [ATP]/[ADP] ratio. Similar arguments show the impor- tance of maintaining appropriate [NADH]/[NAD H11001 ] and [NADPH]/[NADP H11001 ] ratios. AMP concentration is a much more sensitive indi- cator of a cell’s energetic state than is ATP. Normally cells have a far higher concentration of ATP (5 to 10 mM) than of AMP (H110210.1 mM). When some process (say, mus- cle contraction) consumes ATP, AMP is produced in two steps. First, hydrolysis of ATP produces ADP, then the reaction catalyzed by adenylate kinase produces AMP: 2 ADP 88n AMP H11001 ATP [ADP][glucose 6-phosphate] H5007H5007H5007H5007 [ATP][glucose] [ADP] eq [glucose 6-phosphate] eq H5007H5007H5007H5007 [ATP] eq [glucose] eq 15.2 Regulation of Metabolic Pathways 571 8885d_c15_560-600 2/26/04 9:04 AM Page 571 mac76 mac76:385_reb: If [ATP] drops by 10%, producing ADP and AMP in the same amounts, the relative change in [AMP] is much greater (Table 15–1). It is not surprising, therefore, that many regulatory processes hinge on changes in [AMP]. One important mediator of regulation by AMP is AMP- dependent protein kinase (AMPK), which responds to an increase in [AMP] by phosphorylating key proteins, thereby regulating their activities. The rise in [AMP] may be caused by a reduced nutrient supply or increased ex- ercise. The action of AMPK (not to be confused with the cyclic AMP–dependent protein kinase; see Section 15.4) increases glucose transport and activates glycoly- sis and fatty acid oxidation, while suppressing energy- requiring processes such as the synthesis of fatty acids, cholesterol, and protein. We discuss this enzyme fur- ther, and the detailed mechanisms by which it effects these changes, in Chapter 23. In addition to the cofactors ATP, NADH, and NADPH, hundreds of metabolic intermediates also must be present at appropriate concentrations in the cell. For example, the glycolytic intermediates dihydroxyacetone phosphate and 3-phosphoglycerate are precursors of tri- acylglycerols and serine, respectively. When these prod- ucts are needed, the rate of glycolysis must be adjusted to provide them without reducing the glycolytic pro- duction of ATP. Priorities at the organismal level have also driven the evolution of regulatory mechanisms. In mammals, the brain has virtually no stored source of energy, de- pending instead on a constant supply of glucose from the blood. If glucose in the blood drops from its normal concentration of 4 to 5 mM to half that level, mental con- fusion results, and a fivefold reduction in blood glucose can lead to coma and death. To buffer against changes in blood glucose concentration, release of the hormones insulin and glucagon, elicited by high or low blood glu- cose, respectively, triggers metabolic changes that tend to return the blood glucose concentration to normal. Other selective pressures must also have operated throughout evolution, selecting for regulatory mecha- nisms that 1. maximize the efficiency of fuel utilization by preventing the simultaneous operation of pathways in opposite directions (such as glycolysis and gluconeogenesis); 2. partition metabolites appropriately between alternative pathways (such as glycolysis and the pentose phosphate pathway); 3. draw on the fuel best suited for the immediate needs of the organism (glucose, fatty acids, glycogen, or amino acids); and 4. shut down biosynthetic pathways when their products accumulate. The importance of effective metabolic regulation is clear from the consequences of failed regulation: in many cases, serious disease (as described in Box 15–1, for example). Regulatory Enzymes Respond to Changes in Metabolite Concentration Flux through a biochemical pathway depends on the ac- tivities of the enzymes that catalyze each reaction in that pathway. For some steps, the reaction is close to equilibrium within the cell (Fig. 15–12). The net flow of metabolites through these steps is the small difference between the rates of the forward and reverse reactions, rates that are very similar when the reaction is near equilibrium. Small changes in substrate or product con- centration can produce large changes in the net rate, and can even change the direction of the net flow. We can identify these near-equilibrium reactions in a cell by comparing the mass action ratio, Q, with the equilib- rium constant for the reaction, KH11032 eq . Recall that for the reaction A H11001 B n C H11001 D, Q H11005 [C][D]/[A][B]. When Q and KH11032 eq are within a few orders of magnitude, the reac- tion is near equilibrium. This is the case for six of the ten reactions in the glycolytic pathway (Table 15–2). Other reactions are far from equilibrium in the cell. For example, KH11032 eq for the phosphofructokinase-1 (PFK-1) reaction in glycolysis is about 1,000, but Q ([fructose 1,6-bisphosphate] [ADP] / [fructose 6-phosphate] [ATP]) in a typical cell in the steady state is about 0.1 (Table 15–2). It is because the reaction is so far from equilib- rium that the process is exergonic under cellular con- Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen572 Concentration before Concentration after Adenine ATP depletion ATP depletion nucleotide (mM)(mM) Relative change ATP 5.0 4.5 10% ADP 1.0 1.0 0 AMP 0.1 0.6 600% TABLE 15–1 Relative Changes in [ATP] and [AMP] When ATP Is Consumed 8885d_c15_560-600 2/26/04 9:04 AM Page 572 mac76 mac76:385_reb: ditions and tends to go in the forward direction. The re- action is held far from equilibrium because, under pre- vailing cellular conditions of substrate, product, and ef- fector concentrations, the rate of conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is limited by the activity of PFK-1, which is itself limited by the num- ber of PFK-1 molecules present and by the actions of effectors. Thus the net forward rate of the enzyme- catalyzed reaction is equal to the net flow of glycolytic intermediates through other steps in the pathway, and the reverse flow through PFK-1 remains near zero. The cell cannot allow reactions with large equilib- rium constants to reach equilibrium. If [fructose 6-phos- phate], [ATP], and [ADP] in the cell were held at their usual level (low millimolar) and the PFK-1 reaction were allowed to reach equilibrium by an increase in [fructose 1,6-bisphosphate], the concentration of fructose 1,6- bisphosphate would rise into the molar range, wreaking osmotic havoc on the cell. Consider another case: if the reaction ATP n ADP H11001 P i were allowed to approach equilibrium in the cell, the actual free-energy change (H9004GH11032) for that reaction (see Box 13–1) would approach zero, and ATP would lose the high phosphoryl group transfer potential that makes it valuable to the cell as an energy source. It is therefore essential that enzymes catalyzing ATP breakdown and other highly exergonic reactions in a cell be sensitive to regulation, so that when metabolic changes are forced by external circum- stances, the flow through these enzymes will be adjusted to ensure that [ATP] remains far above its equilibrium level. When such metabolic changes occur, enzymatic activities in all interconnected pathways adjust to keep these critical steps away from equilibrium. Thus, not surprisingly, many enzymes that catalyze highly exer- gonic reactions (such as PFK-1) are subject to a vari- ety of subtle regulatory mechanisms. The multiplicity of these adjustments is so great that we cannot predict by examining the properties of any one enzyme in a path- way whether that enzyme has a strong influence on net 15.2 Regulation of Metabolic Pathways 573 V H11005 10.01 V H11005 200 V H11005 500 1 23 V H11005 0.01 V H11005 190 V H11005 490 10net rate: 10 10 ABCD FIGURE 15–12 Near-equilibrium and nonequilibrium steps in a metabolic pathway. Steps 2 and 3 of this pathway are near equilib- rium in the cell; their forward rates are only slightly greater than their reverse rates, so the net forward rates (10) are relatively low and the free-energy change H9004GH11032 for each step is close to zero. An increase in the intracellular concentration of metabolite C or D can reverse the direction of these steps. Step 1 is maintained in the cell far from equi- librium; its forward rate greatly exceeds its reverse rate. The net rate of step 1 (10) is much larger than the reverse rate (0.09) and is iden- tical to the net rates of steps 2 and 3 when the pathway is operating in the steady state. Step 1 has a large, negative H9004GH11032. Reaction near ?GH11032 Mass action ratio, Q equilibrium ?GH11032o (kJ/mol) Enzyme KH11032 eq Liver Heart in vivo?* (kJ/mol) in heart Hexokinase 1.2 H11003 10 3 3.2 H11003 10 H110022 3.8 H11003 10 H110022 No H1100217 H1100227 PFK-1 1.0 H11003 10 3 3.9 H11003 10 H110022 3.3 H11003 10 H110022 No H1100214.2 H1100223 Aldolase 1.0 H11003 10 H110024 1.2 H11003 10 H110026 3.9 H11003 10 H110026 Yes H1100123.8 H110026.0 Triose phosphate isomerase 1.4 H11003 10 H110022 — 2.4 H11003 10 H110021 Yes H110017.5 H110013.8 Glyceraldehyde 3-phosphate dehydrogenase H11001 phosphoglycerate kinase 1.2 H11003 10 3 3.6 H11003 10 2 9.0 Yes H1100213 H110013.5 Phosphoglycerate mutase 1 H11003 10 H110021 3.1 H11003 10 H110021 1.2 H11003 10 H110021 Yes H110014.4 H110010.6 Enolase 3 2.9 1.4 Yes H110023.2 H110020.5 Pyruvate kinase 1.2 H11003 10 4 3.7 H11003 10 H110021 40 No H1100231.0 H1100217 Phosphoglucose isomerase 4 H11003 10 H110021 3.1 H11003 10 H110021 2.4 H11003 10 H110021 Yes H110012.2 H110021.4 Pyruvate carboxylase H11001 PEP carboxykinase 7 3.1 H11003 10 H110023 —NoH110025.0 H1100222.8 Glucose 6-phosphatase 8.5 H11003 10 2 1.2 H11003 10 2 —YesH1100217.3 H110025.0 TABLE 15–2 *For simplicity, any reaction for which the absolute value of the calculated H9004GH11032 is less than 6 is considered near equilibrium. Source: KH11032 eq and Q from Newsholme, E.A. & Start, C. (1973) Regulation in Metabolism, Wiley Press, New York, pp. 97, 263 H9004GH11032 and H9004GH11032o were calculated from these data. Equilibrium Constants, Mass Action Coefficients, and Free-Energy Changes for Enzymes of Carbohydrate Metabolism 8885d_c15_560-600 2/26/04 9:04 AM Page 573 mac76 mac76:385_reb: flow through the entire pathway. This complex problem can be approached by metabolic control analysis, as de- scribed in Section 15.5. Enzyme Activity Can Be Altered in Several Ways The activity of an enzyme can be modulated by changes in the number of enzyme molecules in the cell or by changes in the catalytic activity of each enzyme mole- cule already present, for example through allosteric regulation or covalent alteration (Fig. 15–13). The num- ber of enzyme molecules in the cell is a function of the rates of synthesis and degradation, both of which, for many enzymes, are tightly controlled. The rate of syn- thesis of a protein can be adjusted by the production or alteration (in response to some outside signal) of a tran- scription factor, a protein that binds to a regulatory re- gion of DNA adjacent to the gene in question and in- creases the likelihood of its transcription into mRNA (Chapter 28). The stability of mRNAs—their resistance to degradation by a ribonuclease—varies, so the amount of a given mRNA in the cell is a function of its rates of synthesis and degradation (Chapter 26). Finally, the rate at which an mRNA is translated on the ribosome depends on several factors, described in detail in Chapter 27. The rate of protein degradation also differs from en- zyme to enzyme and depends on the conditions in the cell; protein half-lives vary from a few minutes to many days. Some proteins are tagged for degradation in pro- teasomes (discussed in Chapter 28) by the covalent at- tachment of ubiquitin (recall the case of cyclin; see Fig. 12–44). Some proteins are synthesized as inactive forms, or proenzymes, that become active only when a proteolytic event removes an inhibitory sequence in the proenzyme. As a result of these several mechanisms of regulat- ing enzyme level, cells can change their complement of enzymes in response to changes in metabolic circum- stances. In vertebrates, liver is the most adaptable tis- sue; a change in diet from high carbohydrate to high lipid, for example, affects the transcription into mRNA of hundreds of genes and thus the levels of hundreds of proteins. These global changes in gene expression can be quantified by the use of DNA microarrays (see Fig. 9–22) or two-dimensional gel electrophoresis (see Fig. 3–22) to display the protein complement of a tissue. Both techniques provide great insights into metabolic regulation. Changes in the number of molecules of an enzyme are generally relatively slow, occurring over seconds to hours. Covalent modifications of existing proteins are faster, taking seconds to minutes. Various types of cova- lent modifications are known, such as adenylylation, methylation, or attachment of lipids (p. 228). By far the most common type is phosphorylation and dephospho- rylation (Fig. 15–14); up to half of a eukaryotic cell’s proteins are targets of phosphorylation under some cir- cumstances. Phosphorylation may alter the electrostatic features of the active site, cause movement of an in- hibitory region of the protein out of the active site, alter the protein’s interaction with other proteins, or force con- formational changes that translate into changes in V max or K m . For covalent modification to be useful in regula- tion, the cell must be able to restore the altered enzyme to its original condition. A family of phosphoprotein phos- phatases, at least some of which are themselves under regulation, catalyze dephosphorylation of proteins that have been phosphorylated by protein kinases. Alteration of the number of enzyme molecules and covalent modifications are generally triggered by some signal from outside the cell—a hormone or growth fac- tor, for example—and result in a change of metabolite Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen574 Ser/Thr/Tyr Ser/Thr/Tyr ATP ADP OH Protein substrate P i phosphoprotein phosphatase H 2 O O PO O – O – protein kinase FIGURE 15–14 Protein phosphorylation and dephosphorylation. Pro- tein kinases transfer a phosphoryl group from ATP to a Ser, Thr, or Tyr residue in a protein. Protein phosphatases remove the phosphoryl group as P i . DNA mRNA Enzyme Amino acids Association with regulatory protein Sequestration (compartmentation) Allosteric regulation Covalent modification Nucleotides transcription translation turnover turnover FIGURE 15–13 Factors that determine the activity of an enzyme. Blue arrows represent processes that determine the number of enzyme molecules in the cell; red arrows show factors that determine the enzy- matic activity of an existing enzyme molecule. Each arrow represents a point at which regulation can occur. 8885d_c15_560-600 2/26/04 9:04 AM Page 574 mac76 mac76:385_reb: flux through one or more pathways. In contrast, the very rapid (milliseconds) allosteric changes in enzyme activ- ity are generally triggered locally, by changes in the level of a metabolite within the cell. The allosteric effector may be a substrate of the affected pathway (glucose for glycolysis, for example), a product of a pathway (ATP from glycolysis), or a key metabolite or cofactor (such as NADH) that indicates the cell’s metabolic state. A sin- gle enzyme is commonly regulated in several ways—for example, by modulation of its synthesis, by covalent al- teration, and by allosteric effectors. Yet another way to alter the effective activity of an enzyme is to change the accessibility of its substrate. The hexokinase of muscle cannot act on glucose until the sugar enters the myocyte from the blood, and the rate at which it enters depends on the activity of glu- cose transporters in the plasma membrane. Within cells, membrane-bounded compartments segregate certain enzymes and enzyme systems, and the transport of sub- strate into these compartments may be the limiting fac- tor in enzyme action. In the discussion that follows, it is useful to think of changes in enzymatic activity as serving two distinct though complementary roles. We use the term meta- bolic regulation to refer to processes that serve to maintain homeostasis at the molecular level—to hold some cellular parameter (concentration of a metabolite, for example) at a steady level over time, even as the flow of metabolites through the pathway changes. The term metabolic control refers to a process that leads to a change in the output of a metabolic pathway over time, in response to some outside signal or change in circumstances. The distinction, although useful, is not always easy to make. SUMMARY 15.2 Regulation of Metabolic Pathways ■ In a metabolically active cell in a steady state, intermediates are formed and consumed at equal rates. When a perturbation alters the rate of formation or consumption of a metabolite, compensating changes in enzyme activities return the system to the steady state. ■ Regulatory mechanisms maintain nearly constant levels of key metabolites such as ATP and NADH in cells and glucose in the blood, while matching the use or storage of glycogen to the organism’s changing needs. ■ In multistep processes such as glycolysis, certain reactions are essentially at equilibrium in the steady state; the rates of these substrate-limited reactions rise and fall with substrate concentration. Other reactions are far from equilibrium; their rates are too slow to produce instant equilibration of substrate and product. These enzyme-limited reactions are often highly exergonic and therefore metabolically irreversible, and the enzymes that catalyze them are commonly the points at which flux through the pathway is regulated. ■ The activity of an enzyme can be regulated by changing the rate of its synthesis or degradation, by allosteric or covalent alteration of existing enzyme molecules, or by separating the enzyme from its substrate in subcellular compartments. ■ Fast metabolic adjustments (on the time scale of seconds or less) at the intracellular level are generally allosteric. The effects of hormones and growth factors are generally slower (seconds to hours) and are commonly achieved by covalent modification or changes in enzyme synthesis. 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis In mammals, gluconeogenesis occurs primarily in the liver, where its role is to provide glucose for export to other tissues when glycogen stores are exhausted. Glu- coneogenesis employs most of the enzymes that act in glycolysis, but it is not simply the reversal of glycolysis. Seven of the glycolytic reactions are freely reversible, and the enzymes that catalyze these reactions also func- tion in gluconeogenesis (Fig. 15–15). Three reactions of glycolysis are so exergonic as to be essentially irre- versible: those catalyzed by hexokinase, PFK-1, and pyruvate kinase. Notice in Table 15–2 that all three re- actions have a large, negative H9004GH11032. Gluconeogenesis uses detours around each of these irreversible steps; for example, the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate is catalyzed by fructose 1,6- bisphosphatase (FBPase-1; Fig. 15–15). Note that each of these bypass reactions also has a large, negative H9004GH11032. At each of the three points where glycolytic reac- tions are bypassed by alternative, gluconeogenic reac- tions, simultaneous operation of both pathways would consume ATP without accomplishing any chemical or biological work. For example, PFK-1 and FBPase-1 cat- alyze opposing reactions: PFK-1 ATP H11001 fructose 6-phosphate 8888n ADP H11001 fructose 1,6-bisphosphate FBPase-1 Fructose 1,6-bisphosphate H11001 H 2 O 888888n fructose 6-phosphate H11001 P i The sum of these two reactions is ATP H11001 H 2 O 88n ADP H11001 P i H11001 heat that is, hydrolysis of ATP without any useful metabolic work being done. Clearly, if these two reactions were 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 575 8885d_c15_560-600 2/26/04 9:04 AM Page 575 mac76 mac76:385_reb: allowed to proceed simultaneously at a high rate in the same cell, a large amount of chemical energy would be dissipated as heat. This uneconomical process has been called a futile cycle. However, as we shall see later, such cycles may provide advantages for controlling pathways, and the term substrate cycle is a better de- scription. Similar substrate cycles also occur with the other two sets of bypass reactions of gluconeogenesis (Fig. 15–15). We begin our examination of the coordinated regu- lation of glycolysis and gluconeogenesis by considering the regulatory patterns seen at the three main control points of glycolysis. We then look at the regulation of the enzymes of gluconeogenesis, leading to a consider- ation of how the regulation of both pathways is tightly, reciprocally coordinated. Hexokinase Isozymes of Muscle and Liver Are Affected Differently by Their Product, Glucose 6-Phosphate Hexokinase, which catalyzes the entry of free glucose into the glycolytic pathway, is a regulatory enzyme. There are four isozymes (designated I to IV), encoded Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen576 hexokinase Glucose Glucose 6-phosphate glucose 6-phosphatase phosphohexose isomerase phospho- fructokinase-1 Fructose 6-phosphate Fructose 1,6-bisphosphate (2) Glyceraldehyde 3-phosphate fructose 1,6-bisphosphatase (2) 1,3-Bisphosphoglycerate (2) 3-Phosphoglycerate (2) 2-Phosphoglycerate (2) Phosphoenolpyruvate (2) Pyruvate (2) Oxaloacetate pyruvate carboxylase PEP carboxykinase pyruvate kinase Dihydroxyacetone phosphate Dihydroxyacetone phosphate aldolase triose phosphate isomerase triose phosphate isomerase glyceraldehyde phosphate dehydrogenase phosphoglycerate kinase phosphoglycerate mutase enolase Glycolysis Gluconeogenesis FIGURE 15–15 Glycolysis and gluconeogenesis. Opposing path- ways of glycolysis (pink) and gluconeogenesis (blue) in rat liver. Three steps are catalyzed by different enzymes in gluconeogenesis (the “bypass reactions”) and glycolysis; seven steps are catalyzed by the same enzymes in the two pathways. Cofactors have been omitted for simplicity. 8885d_c15_560-600 2/26/04 9:04 AM Page 576 mac76 mac76:385_reb: by four different genes. Isozymes are different proteins that catalyze the same reaction (Box 15–2). The pre- dominant hexokinase isozyme of myocytes (hexokinase II) has a high affinity for glucose—it is half-saturated at about 0.1 mM. Because glucose entering myocytes from the blood (where the glucose concentration is 4 to 5 mM) produces an intracellular glucose concentration high enough to saturate hexokinase II, the enzyme normally acts at or near its maximal rate. Muscle hexokinases I and II are allosterically inhibited by their product, glu- cose 6-phosphate, so whenever the cellular concentra- tion of glucose 6-phosphate rises above its normal level, these isozymes are temporarily and reversibly inhibited, bringing the rate of glucose 6-phosphate formation into balance with the rate of its utilization and reestablishing the steady state. The different hexokinase isozymes of liver and mus- cle reflect the different roles of these organs in carbo- hydrate metabolism: muscle consumes glucose, using it for energy production, whereas liver maintains blood glu- cose homeostasis by removing or producing glucose, de- pending on the prevailing glucose concentration. The 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 577 BOX 15–2 WORKING IN BIOCHEMISTRY Isozymes: Different Proteins That Catalyze the Same Reaction The four forms of hexokinase found in mammalian tis- sues are but one example of a common biological sit- uation: the same reaction catalyzed by two or more different molecular forms of an enzyme. These multi- ple forms, called isozymes or isoenzymes, may occur in the same species, in the same tissue, or even in the same cell. The different forms of the enzyme gener- ally differ in kinetic or regulatory properties, in the cofactor they use (NADH or NADPH for dehydroge- nase isozymes, for example), or in their subcellular distribution (soluble or membrane-bound). Isozymes may have similar, but not identical, amino acid se- quences, and in many cases they clearly share a com- mon evolutionary origin. One of the first enzymes found to have isozymes was lactate dehydrogenase (LDH) (p. 538), which, in vertebrate tissues, exists as at least five different isozymes separable by electrophoresis. All LDH isozymes contain four polypeptide chains (each of M r 33,500), each type containing a different ratio of two kinds of polypeptides. The M (for muscle) chain and the H (for heart) chain are encoded by two different genes. In skeletal muscle the predominant isozyme con- tains four M chains, and in heart the predominant isozyme contains four H chains. Other tissues have some combination of the five possible types of LDH isozymes: Type Composition Location LDH 1 HHHH Heart and erythrocyte LDH 2 HHHM Heart and erythrocyte LDH 3 HHMM Brain and kidney LDH 4 HMMM Skeletal muscle and liver LDH 5 MMMM Skeletal muscle and liver These differences in the isozyme content of tis- sues can be used to assess the timing and ex- tent of heart damage due to myocardial infarction (heart attack). Damage to heart tissue results in the release of heart LDH into the blood. Shortly after a heart attack, the blood level of total LDH increases, and there is more LDH 2 than LDH 1 . After 12 hours the amounts of LDH 1 and LDH 2 are very similar, and af- ter 24 hours there is more LDH 1 than LDH 2 . This switch in the LDH 1 /LDH 2 ratio, combined with in- creased concentrations in the blood of another heart enzyme, creatine kinase, is very strong evidence of a recent myocardial infarction. ■ The different LDH isozymes have significantly dif- ferent values of V max and K M , particularly for pyru- vate. The properties of LDH 4 favor rapid reduction of very low concentrations of pyruvate to lactate in skele- tal muscle, whereas those of isozyme LDH 1 favor rapid oxidation of lactate to pyruvate in the heart. In general, the distribution of different isozymes of a given enzyme reflects at least four factors: 1. Different metabolic patterns in different or- gans. For glycogen phosphorylase, the isozymes in skeletal muscle and liver have different regula- tory properties, reflecting the different roles of glycogen breakdown in these two tissues. 2. Different locations and metabolic roles for isozymes in the same cell. The isocitrate dehy- drogenase isozymes of the cytosol and the mito- chondrion are an example (Chapter 16). 3. Different stages of development in embryonic or fetal tissues and in adult tissues. For exam- ple, the fetal liver has a characteristic isozyme dis- tribution of LDH, which changes as the organ de- velops into its adult form. Some enzymes of glucose catabolism in malignant (cancer) cells oc- cur as their fetal, not adult, isozymes. 4. Different responses of isozymes to allosteric modulators. This difference is useful in fine-tun- ing metabolic rates. Hexokinase IV (glucokinase) of liver and the hexokinase isozymes of other tis- sues differ in their sensitivity to inhibition by glu- cose 6-phosphate. 8885d_c15_560-600 2/26/04 9:04 AM Page 577 mac76 mac76:385_reb: predominant hexokinase isozyme of liver is hexokinase IV (glucokinase), which differs in three important respects from hexokinases I–III of muscle. First, the glucose concentration at which hexokinase IV is half- saturated (about 10 mM) is higher than the usual con- centration of glucose in the blood. Because an efficient glucose transporter in hepatocytes (GLUT2; see Fig. 11–31) rapidly equilibrates the glucose concentrations in cytosol and blood, the high K m of hexokinase IV allows its direct regulation by the level of blood glucose (Fig. 15–16). When the blood glucose concentration is high, as it is after a meal rich in carbohydrates, excess glucose is transported into hepatocytes, where hexokinase IV converts it to glucose 6-phosphate. Because hexokinase IV is not saturated at 10 mM glucose, its activity con- tinues to increase as the glucose concentration rises to 10 mM or more. Second, hexokinase IV is subject to inhibition by the reversible binding of a regulatory protein specific to liver (Fig. 15–17). The binding is much tighter in the pres- ence of the allosteric effector fructose 6-phosphate. Glu- cose competes with fructose 6-phosphate for binding and causes dissociation of the regulatory protein from hexokinase IV, relieving the inhibition. Immediately af- ter a carbohydrate-rich meal, when blood glucose is high, glucose enters the hepatocyte via GLUT2 and ac- tivates hexokinase IV by this mechanism. During a fast, when blood glucose drops below 5 mM, fructose 6- phosphate triggers the inhibition of hexokinase IV by the regulatory protein, so the liver does not compete with other organs for the scarce glucose. The mecha- nism of inhibition by the regulatory protein is interest- ing: the protein anchors hexokinase IV inside the nu- cleus, where it is segregated from the other enzymes of glycolysis in the cytosol (Fig. 15–17). When the glucose concentration in the cell rises, it equilibrates with glu- cose in the nucleus by transport through the nuclear pores. Glucose causes dissociation of the regulatory pro- tein, and hexokinase IV enters the cytosol and begins to phosphorylate glucose. Third, hexokinase IV is not inhibited by glucose 6- phosphate, and it can therefore continue to operate when the accumulation of glucose 6-phosphate com- pletely inhibits hexokinases I–III. Phosphofructokinase-1 Is under Complex Allosteric Regulation As we have noted, glucose 6-phosphate can flow either into glycolysis or through any of several other pathways, including glycogen synthesis and the pentose phosphate pathway. The metabolically irreversible reaction cat- alyzed by PFK-1 is the step that commits glucose to gly- colysis. In addition to its substrate-binding sites, this Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen578 1.0 0510 Glucose concentration (mM) 15 20 Relative enzyme activity Hexokinase IV (glucokinase) Hexokinase I Glucose 6-phosphate Fructose 6-phosphate Hexokinase IV Hexokinase IV Glucose GLUT2 Plasma membrane Cytosol Nucleus Regulator protein Glucose Capillary FIGURE 15–16 Comparison of the kinetic properties of hexokinase IV (glucokinase) and hexokinase I. Note the sigmoidicity for hexoki- nase IV and the much lower K m for hexokinase I. When blood glu- cose rises above 5 mM, hexokinase IV activity increases, but hexoki- nase I is already operating near V max at 5 mM glucose and cannot respond to an increase in glucose concentration. Hexokinase I, II, and III have similar kinetic properties. FIGURE 15–17 Regulation of hexoki- nase IV (glucokinase) by sequestration in the nucleus. The protein inhibitor of hexokinase IV is a nuclear binding protein that draws hexokinase IV into the nucleus when the fructose 6- 0phosphate concentration in liver is high and releases it to the cytosol when the glucose concentration is high. 8885d_c15_560-600 2/26/04 9:04 AM Page 578 mac76 mac76:385_reb: complex enzyme has several regulatory sites at which allosteric activators or inhibitors bind. ATP is not only a substrate for PFK-1 but also an end product of the glycolytic pathway. When high cel- lular [ATP] signals that ATP is being produced faster than it is being consumed, ATP inhibits PFK-1 by bind- ing to an allosteric site and lowering the affinity of the enzyme for fructose 6-phosphate (Fig. 15–18). ADP and AMP, which increase in concentration as consumption of ATP outpaces production, act allosterically to relieve this inhibition by ATP. These effects combine to pro- duce higher enzyme activity when ADP or AMP accu- mulates and lower activity when ATP accumulates. Citrate (the ionized form of citric acid), a key in- termediate in the aerobic oxidation of pyruvate, fatty acids, and amino acids, also serves as an allosteric reg- ulator of PFK-1; high citrate concentration increases the inhibitory effect of ATP, further reducing the flow of glu- cose through glycolysis. In this case, as in several oth- ers encountered later, citrate serves as an intracellular signal that the cell is meeting its current needs for energy-yielding metabolism by the oxidation of fats and proteins. The most significant allosteric regulator of PFK-1 is fructose 2,6-bisphosphate, which strongly activates the enzyme. We return to this role of fructose 2,6- bisphosphate later. Pyruvate Kinase Is Allosterically Inhibited by ATP At least three isozymes of pyruvate kinase are found in vertebrates, differing in their tissue distribution and their response to modulators. High concentrations of ATP, acetyl-CoA, and long-chain fatty acids (signs of abundant energy supply) allosterically inhibit all isozymes of pyru- vate kinase (Fig. 15–19). The liver isozyme (L form), but not the muscle isozyme (M form), is subject to further regulation by phosphorylation. When low blood glucose causes glucagon release, cAMP-dependent protein kinase phosphorylates the L isozyme of pyruvate kinase, inacti- vating it. This slows the use of glucose as a fuel in liver, sparing it for export to the brain and other organs. In muscle, the effect of increased [cAMP] is quite different. In response to epinephrine, cAMP activates glycogen breakdown and glycolysis and provides the fuel needed for the fight-or-flight response. 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 579 (a) Fructose 6- phosphate H11001 ATP Fructose 1,6- bisphosphate citrate ATP AMP, ADP fructose 2,6- bisphosphate H11001 ADP (c) PFK-1 activity [Fructose 6-phosphate] (b) High [ATP] Low [ATP] FIGURE 15–18 Phosphofructokinase-1 (PFK-1) and its regulation. (a) Ribbon diagram of E. coli phosphofructokinase-1, showing two of its four identical subunits (PDB ID 1PFK). Each subunit has its own cat- alytic site, where ADP (blue) and fructose 1,6-bisphosphate (yellow) are almost in contact, and its own binding sites for the allosteric regulator ADP (blue), located at the interface between subunits. (b) Allosteric regulation of muscle PFK-1 by ATP, shown by a substrate-activity curve. At low concentrations of ATP, the K 0.5 for fructose 6-phosphate is rel- atively low, enabling the enzyme to function at a high rate at relatively low concentrations of fructose 6-phosphate. (Recall from Chapter 6 that K 0.5 or K m is equivalent to the substrate concentration at which half-maximal enzyme activity occurs.) When the concentration of ATP is high, K 0.5 for fructose 6-phosphate is greatly increased, as indicated by the sigmoid relationship between substrate concentration and en- zyme activity. (c) Summary of the regulators affecting PFK-1 activity. 8885d_c15_560-600 2/26/04 9:04 AM Page 579 mac76 mac76:385_reb: Gluconeogenesis Is Regulated at Several Steps In the pathway leading from pyruvate to glucose, the first control point determines the fate of pyruvate in the mitochondrion. Pyruvate can be converted either to acetyl-CoA (by the pyruvate dehydrogenase complex; Chapter 16) to fuel the citric acid cycle, or to oxalo- acetate (by pyruvate carboxylase) to start the process of gluconeogenesis (Fig. 15–20). When fatty acids are readily available as fuels, their breakdown in liver mito- chondria yields acetyl-CoA, a signal that further oxida- tion of glucose for fuel is not necessary. Acetyl-CoA is a positive allosteric modulator of pyruvate carboxylase and a negative modulator of pyruvate dehydrogenase, through stimulation of a protein kinase that inactivates the dehydrogenase. When the cell’s energetic needs are being met, oxidative phosphorylation slows, NADH rises relative to NAD H11001 and inhibits the citric acid cycle, and acetyl-CoA accumulates. The increased concentration of acetyl-CoA inhibits the pyruvate dehydrogenase complex, slowing the formation of acetyl-CoA from pyruvate, and stimulates gluconeogenesis by activating pyruvate carboxylase, allowing excess pyruvate to be converted to glucose. The second control point in gluconeogenesis is the reaction catalyzed by FBPase-1 (Fig. 15–21), which is strongly inhibited by AMP. The corresponding glycolytic enzyme, PFK-1, is stimulated by AMP and ADP but in- hibited by citrate and ATP. Thus these opposing steps Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen580 FIGURE 15–19 Regulation of pyruvate kinase. The enzyme is al- losterically inhibited by ATP, acetyl-CoA, and long-chain fatty acids (all signs of an abundant energy supply), and the accumulation of fruc- tose 1,6-bisphosphate triggers its activation. Accumulation of alanine, which can be synthesized from pyruvate in one step, allosterically in- hibits pyruvate kinase, slowing the production of pyruvate by glycol- ysis. The liver isozyme (L form) is also regulated hormonally; glucagon activates cAMP-dependent protein kinase (PKA; see Fig. 15–25), which phosphorylates the pyruvate kinase L isozyme, inactivating it. When the glucagon level drops, a protein phosphatase (PP) dephosphory- lates pyruvate kinase, activating it. This mechanism prevents the liver from consuming glucose by glycolysis when the blood glucose con- centration is low; instead, liver exports glucose. The muscle isozyme (M form) is not affected by this phosphorylation mechanism. Liver only All other glycolytic tissues glucagon ADP ADP ATP ATP ATP, acetyl-CoA, long-chain fatty acids PKA P Pyruvate kinase L/M Pyruvate kinase L (inactive) H 2 O PP P i PEP Pyruvate transamination Alanine F16BP 6 steps Oxaloacetate pyruvate carboxylase Gluconeogenesis Glucose Pyruvate pyruvate dehydrogenase complex Acetyl-CoA Citric acid cycle CO 2 Energy FIGURE 15–20 Two alternative fates for pyruvate. Pyruvate can be converted to glucose and glycogen via gluconeogenesis or oxidized to acetyl-CoA for energy production. The first enzyme in each path is regulated allosterically; acetyl-CoA, produced either by fatty acid oxidation or by the pyruvate dehydrogenase complex, stimulates pyruvate carboxylase and inhibits pyruvate dehydrogenase. 8885d_c15_560-600 2/26/04 9:04 AM Page 580 mac76 mac76:385_reb: in the two pathways are regulated in a coordinated and reciprocal manner. In general, when sufficient concen- trations of acetyl-CoA or citrate (the product of acetyl- CoA condensation with oxaloacetate) are present, or when a high proportion of the cell’s adenylate is in the form of ATP, gluconeogenesis is favored. AMP promotes glycogen degradation and glycolysis by activating glyco- gen phosphorylase (via activation of phosphorylase ki- nase) and stimulating the activity of PFK-1. All the regulatory actions discussed here are trig- gered by changes inside the cell and are mediated by very rapid, instantly reversible, allosteric mechanisms. Another set of regulatory processes is triggered from outside the cell by the hormones insulin and glucagon, which signal too much or too little glucose in the blood, respectively, or by epinephrine, which signals the im- pending need for fuel for a fight-or-flight response. These hormonal signals bring about covalent modification (phosphorylation or dephosphorylation) of target pro- teins inside the cell; this takes place on a somewhat longer time scale than the internally driven allosteric mecha- nisms—seconds or minutes, rather than milliseconds. Fructose 2,6-Bisphosphate Is a Potent Regulator of Glycolysis and Gluconeogenesis The special role of liver in maintaining a constant blood glucose level requires additional regulatory mechanisms to coordinate glucose production and consumption. When the blood glucose level decreases, the hormone glucagon signals the liver to produce and release more glucose and to stop consuming it for its own needs. One source of glucose is glycogen stored in the liver; another source is gluconeogenesis. The hormonal regulation of glycolysis and gluco- neogenesis is mediated by fructose 2,6-bisphosphate, an allosteric effector for the enzymes PFK-1 and FBPase-1 (Fig. 15–22): When fructose 2,6-bisphosphate binds to its allosteric site on PFK-1, it increases that enzyme’s affinity for its substrate, fructose 6-phosphate, and reduces its affin- ity for the allosteric inhibitors ATP and citrate. At the physiological concentrations of its substrates ATP and fructose 6-phosphate and of its other positive and neg- ative effectors (ATP, AMP, citrate), PFK-1 is virtually inactive in the absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate activates PFK-1 and stimu- lates glycolysis in liver and, at the same time, inhibits FBPase-1, thereby slowing gluconeogenesis. Although structurally related to fructose 1,6- bisphosphate, fructose 2,6-bisphosphate is not an in- termediate in gluconeogenesis or glycolysis; it is a reg- ulator whose cellular level reflects the level of glucagon in the blood, which rises when blood glucose falls. The cellular concentration of fructose 2,6-bisphosphate is set by the relative rates of its formation and breakdown (Fig. 15–23a). It is formed by phosphorylation of fructose 6-phosphate, catalyzed by phosphofructoki- nase-2 (PFK-2), and is broken down by fructose 2,6- bisphosphatase (FBPase-2). (Note that these en- zymes are distinct from PFK-1 and FBPase-1, which catalyze the formation and breakdown, respectively, of fructose 1,6-bisphosphate.) PFK-2 and FBPase-2 are two distinct enzymatic activities of a single, bifunctional protein. The balance of these two activities in the liver, which determines the cellular level of fructose 2,6- bisphosphate, is regulated by glucagon and insulin (Fig. 15–23b). As we saw in Chapter 12 (p. 441), glucagon stimulates the adenylyl cyclase of liver to synthesize 3H11032,5H11032-cyclic AMP (cAMP) from ATP. Then cAMP acti- vates cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the bifunctional pro- tein PFK-2/FBPase-2. Phosphorylation of this protein enhances its FBPase-2 activity and inhibits its PFK-2 activity. Glucagon thereby lowers the cellular level of fructose 2,6-bisphosphate, inhibiting glycolysis and stimulating gluconeogenesis. The resulting production of more glucose enables the liver to replenish blood glu- cose in response to glucagon. Insulin has the opposite effect, stimulating the activity of a phosphoprotein phos- phatase that catalyzes removal of the phosphoryl group from the bifunctional protein PFK-2/FBPase-2, activat- ing its PFK-2 activity, increasing the level of fructose 2,6-bisphosphate, stimulating glycolysis, and inhibiting gluconeogenesis. H CH 2 PO A O HO H11002 O O B H H OH CH 2 OH PO A O O O B O O Fructose 2,6-bisphosphate O O H11002 O H11002 O H11002 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis 581 Fructose 6-phosphate ATP ADP AMP citrate Fructose 1,6-bisphosphate ATP ADP Gluconeogenesis Glycolysis PFK-1 FBPase-1 P i H 2 O FIGURE 15–21 Regulation of fructose 1,6-bisphosphatase-1 (FBPase-1) and phosphofructokinase-1 (PFK-1). The important role of fructose 2,6-bisphosphate in the regulation of this substrate cycle is detailed in subsequent figures. 8885d_c15_581 2/26/04 2:01 PM Page 581 mac76 mac76:385_reb: Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen582 PFK-1 activity (% of V max ) 0 100 [Fructose 6-phosphate] (mM) (a) (c) 80 60 40 20 0.05 0.1 0.2 0.4 0.7 1.0 2.0 FBPase-1 activity (% of V max ) 0 100 [Fructose 1,6-bisphosphate] ( M) (b) 80 60 40 20 50 100 0 0 4.0 H11001F26BP H11002F26BP H11002F26BP H11001F26BP H9262 Fructose 6-phosphate F26BP Fructose 1,6-bisphosphate ATP ADP Gluconeogenesis Glycolysis PFK-1 FBPase-1 P i H 2 O FIGURE 15–22 Role of fructose 2,6-bisphosphate in regulation of glycolysis and gluconeogenesis. Fructose 2,6-bisphosphate (F26BP) has opposite effects on the enzymatic activities of phosphofructoki- nase-1 (PFK-1, a glycolytic enzyme) and fructose 1,6-bisphosphatase (FBPase-1, a gluconeogenic enzyme). (a) PFK-1 activity in the absence of F26BP (blue curve) is half-maximal when the concentration of fructose 6-phosphate is 2 mM (that is, K 0.5 H11005 2 mM). When 0.13 μM F26BP is present (red curve), the K 0.5 for fructose 6-phosphate is only 0.08 mM. Thus F26BP activates PFK-1 by increasing its apparent affin- ity (Fig. 15–18) for fructose 6-phosphate. (b) FBPase-1 activity is in- hibited by as little as 1 H9262M F26BP and is strongly inhibited by 25 H9262M. In the absence of this inhibitor (blue curve) the K 0.5 for fructose 1,6- bisphosphate is 5 H9262M, but in the presence of 25 H9262M F26BP (red curve) the K 0.5 is H11022 70H9262M. Fructose 2,6-bisphosphate also makes FBPase-1 more sensitive to inhibition by another allosteric regulator, AMP. (c) Summary of regulation by F26BP. Fructose 2,6-bisphosphate Fructose 6-phosphate PFK-2 FBPase-2 (a) ATP ADP P i ADP ATP H 2 O P i glucagon [cAMP])( [F26BP] Stimulates glycolysis, inhibits gluconeogenesis Inhibits glycolysis, stimulates gluconeogenesis cAMP-dependent protein kinase phospho- protein phosphatase insulin FBPase-2 (inactive) PFK-2 (active) OH PFK-2 (inactive) FBPase-2 (active) O P O H11002 O H11002 O [F26BP] (b) FIGURE 15–23 Regulation of fructose 2,6-bisphosphate level. (a) The cellular concentration of the regulator fructose 2,6-bisphosphate (F26BP) is determined by the rates of its synthesis by phosphofructo- kinase-2 (PFK-2) and breakdown by fructose 2,6-bisphosphatase (FBPase-2). (b) Both enzymes are part of the same polypeptide chain, and both are regulated, in a reciprocal fashion, by insulin and glucagon. Here and elsewhere, arrows are used to indicate increas- ing (h) and decreasing (g) levels of metabolites. 8885d_c15_582 2/26/04 2:01 PM Page 582 mac76 mac76:385_reb: Are Substrate Cycles Futile? We noted above that substrate cycles (sometimes called futile cycles) occur at several points in the pathways that interconnect glycogen and pyruvate. For reactions such as those catalyzed by PFK-1 and FBPase-1 (Fig. 15–22c) to take place at the same time, each must be exergonic under the conditions prevailing in the cell. The PFK-1 reaction is exergonic because it involves a phosphoryl group transfer from ATP, and the FBPase-1 reaction is exergonic because it entails hydrolysis of a phosphate ester. Because the cycle involves two differ- ent enzymes, not simply one working in both directions, each activity can be regulated separately: fructose 2,6-bisphosphate activates PFK-1, favoring glycolysis, and inhibits FBPase-1, inhibiting gluconeogenesis. The two-enzyme cycle thus provides a means of controlling the direction of net metabolite flow. The apparent en- ergetic disadvantage of the “futile” cycle is evidently outweighed by the advantage of allowing this type of control of pathway direction. Xylulose 5-Phosphate Is a Key Regulator of Carbohydrate and Fat Metabolism Another recently discovered regulatory mechanism also acts by controlling the level of fructose 2,6-bisphos- phate. In the mammalian liver, xylulose 5-phosphate (see Fig. 14–23), a product of the hexose monophos- phate pathway, mediates the increase in glycolysis that follows ingestion of a high-carbohydrate meal. The xylulose 5-phosphate concentration rises as glucose entering the liver is converted to glucose 6-phosphate and enters both the glycolytic and hexose monophos- phate pathways. Xylulose 5-phosphate activates a phos- phoprotein phosphatase, PP2A, that dephosphorylates the bifunctional PFK-2/FBPase-2 enzyme. Dephospho- rylation activates PFK-2 and inhibits FBPase-2, and the resulting rise in [fructose 2,6-bisphosphate] stimulates glycolysis and inhibits gluconeogenesis. The increased glycolysis boosts the production of acetyl-CoA, while the increased flow of hexose through the hexose monophos- phate pathway generates NADPH. Acetyl-CoA and NADPH are the starting materials for fatty acid synthe- sis, which has long been known to increase dramatically in response to intake of a high-carbohydrate meal. Xy- lulose 5-phosphate also increases the synthesis of all the enzymes required for fatty acid synthesis; we shall re- turn to this effect in our discussion of the integration of carbohydrate and lipid metabolism (Chapter 23). SUMMARY 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis ■ Three glycolytic enzymes are subject to allosteric regulation: hexokinase IV, phosphofructokinase-1 (PFK-1), and pyruvate kinase. ■ Hexokinase IV (glucokinase) is sequestered in the nucleus of the hepatocyte, but is released when the cytosolic glucose concentration rises. ■ PFK-1 is allosterically inhibited by ATP and citrate. In most mammalian tissues, including liver, PFK-1 is allosterically activated by fructose 2,6-bisphosphate. ■ Pyruvate kinase is allosterically inhibited by ATP, and the liver isozyme is inhibited by cAMP-dependent phosphorylation. ■ Gluconeogenesis is regulated at the level of pyruvate carboxylase (which is activated by acetyl-CoA) and FBPase-1 (which is inhibited by fructose 2,6-bisphosphate and AMP). ■ To limit futile cycling between glycolysis and gluconeogenesis, the two pathways are under reciprocal allosteric control, mainly achieved by the opposite effects of fructose 2,6- bisphosphate on PFK-1 and FBPase-1. ■ Glucagon or epinephrine decreases [fructose 2,6-bisphosphate]. The hormones do this by raising [cAMP] and bringing about phosphorylation of the bifunctional enzyme that makes and breaks down fructose 2,6- bisphosphate. Phosphorylation inactivates PFK-2 and activates FBPase-2, leading to breakdown of fructose 2,6-bisphosphate. Insulin increases [fructose 2,6-bisphosphate] by activating a phosphoprotein phosphatase that dephosphorylates (activates) PFK-2. 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown As we have seen, the mobilization of stored glycogen is brought about by glycogen phosphorylase, which de- grades glycogen to glucose 1-phosphate (Fig. 15–3). Glycogen phosphorylase provides an especially instruc- tive case of enzyme regulation. It was one of the first known examples of an allosterically regulated enzyme and the first enzyme shown to be controlled by reversible phosphorylation. It was also one of the first allosteric en- zymes for which the detailed three-dimensional struc- tures of the active and inactive forms were revealed by x-ray crystallographic studies. Glycogen phosphorylase also illustrates how isozymes play their tissue-specific roles. Glycogen Phosphorylase Is Regulated Allosterically and Hormonally In the late 1930s, Carl and Gerty Cori (Box 15–1) dis- covered that the glycogen phosphorylase of skeletal muscle exists in two interconvertible forms: glycogen phosphorylase a, which is catalytically active, and 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown 583 8885d_c15_560-600 2/26/04 9:04 AM Page 583 mac76 mac76:385_reb: glycogen phosphorylase b, which is less active (Fig. 15–24). Subsequent studies by Earl Sutherland showed that phosphorylase b predominates in resting muscle, but during vigorous muscular activity the hormone epinephrine triggers phosphorylation of a specific Ser residue in phosphorylase b, converting it to its more active form, phosphorylase a. (Note that glycogen phosphorylase is often referred to simply as phosphorylase—so honored because it was the first phos- phorylase to be discovered; the shortened name has per- sisted in common usage and in the literature.) The enzyme (phosphory- lase b kinase) responsible for activating phosphorylase by transferring a phosphoryl group to its Ser residue is it- self activated by epinephrine or glucagon through a se- ries of steps shown in Figure 15–25. Sutherland discov- ered the second messenger cAMP, which increases in concentration in response to stimulation by epinephrine (in muscle) or glucagon (in liver). Elevated [cAMP] ini- tiates an enzyme cascade, in which a catalyst activates a catalyst, which activates a catalyst. Such cascades al- low for large amplification of the initial signal (see pink boxes in Fig. 15–25). The rise in [cAMP] activates cAMP- dependent protein kinase, also called protein kinase A (PKA). PKA then phosphorylates and activates phos- phorylase b kinase, which catalyzes the phosphoryla- tion of Ser residues in each of the two identical subunits of glycogen phosphorylase, activating it and thus stim- ulating glycogen breakdown. In muscle, this provides fuel for glycolysis to sustain muscle contraction for the fight-or-flight response signaled by epinephrine. In liver, glycogen breakdown counters the low blood glucose sig- naled by glucagon, releasing glucose. These different roles are reflected in subtle differences in the regula- tory mechanisms in muscle and liver. The glycogen phosphorylases of liver and muscle are isozymes, en- coded by different genes and differing in their regula- tory properties. In muscle, superimposed on the regulation of phos- phorylase by covalent modification are two allosteric control mechanisms (Fig. 15–25). Ca 2H11001 , the signal for muscle contraction, binds to and activates phosphory- lase b kinase, promoting conversion of phosphorylase b to the active a form. Ca 2H11001 binds to phosphorylase b ki- nase through its H9254 subunit, which is calmodulin (see Fig. 12–21). AMP, which accumulates in vigorously con- tracting muscle as a result of ATP breakdown, binds to and activates phosphorylase, speeding the release of glucose 1-phosphate from glycogen. When ATP levels are adequate, ATP blocks the allosteric site to which AMP binds, inactivating phosphorylase. When the muscle returns to rest, a second enzyme, phosphorylase a phosphatase, also called phospho- protein phosphatase 1 (PP1), removes the phos- phoryl groups from phosphorylase a, converting it to the less active form, phosphorylase b. Like the enzyme of muscle, the glycogen phospho- rylase of liver is regulated hormonally (by phosphoryla- tion/dephosphorylation) and allosterically. The dephos- phorylated form is essentially inactive. When the blood glucose level is too low, glucagon (acting by the same cascade mechanism shown in Fig. 15–25) activates phosphorylase b kinase, which in turn converts phos- phorylase b to its active a form, initiating the release of glucose into the blood. When blood glucose levels re- turn to normal, glucose enters hepatocytes and binds to an inhibitory allosteric site on phosphorylase a. This binding also produces a conformational change that ex- poses the phosphorylated Ser residues to PP1, which catalyzes their dephosphorylation and inactivates the phosphorylase (Fig. 15–26). The allosteric site for glu- cose allows liver glycogen phosphorylase to act as its own glucose sensor and to respond appropriately to changes in blood glucose. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen584 CH 2 OH CH 2 OH 2ATP2P i 2H 2 O 2ADP phosphorylase b kinase glucagon (liver) epinephrine, [Ca 2+ ], [AMP] (muscle) phosphorylase a phosphatase (PP1) Phosphorylase b (less active) Ser 14 side chain Ser 14 side chain CH 2 CH 2 PP OO Phosphorylase a (active) Earl W. Sutherland, Jr., 1915–1974 FIGURE 15–24 Regulation of muscle glycogen phosphorylase by covalent modification. In the more active form of the enzyme, phos- phorylase a, Ser 14 residues, one on each subunit, are phosphorylated. Phosphorylase a is converted to the less active form, phosphorylase b, by enzymatic loss of these phosphoryl groups, catalyzed by phos- phorylase a phosphatase (PP1). Phosphorylase b can be reconverted (reactivated) to phosphorylase a by the action of phosphorylase b kinase. 8885d_c15_584 2/26/04 2:01 PM Page 584 mac76 mac76:385_reb: 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown 585 FIGURE 15–25 Cascade mechanism of epinephrine and glucagon action. By binding to specific surface receptors, either epinephrine acting on a myocyte (left) or glucagon acting on a hepatocyte (right) acti- vates a GTP-binding protein G sH9251 (see Fig. 12–12). Active G sH9251 triggers a rise in [cAMP], activating PKA. This sets off a cascade of phosphorylations; PKA acti- vates phosphorylase b kinase, which then activates glycogen phosphorylase. Such cascades effect a large amplification of the initial signal; the figures in pink boxes are probably low estimates of the actual increase in number of molecules at each stage of the cascade. The resulting breakdown of glycogen provides glucose, which in the myocyte can supply ATP (via glycolysis) for muscle contraction and in the hepatocyte is released into the blood to counter the low blood glucose. Inactive glycogen phosphorylase b Inactive phosphorylase b kinase Active phosphorylase b kinase Inactive PKA Active PKA Epinephrine G s ATP Hepatocyte Glucagon Cyclic AMP 20× molecules 10× molecules 100× molecules 1,000× molecules 10,000× molecules 10,000× molecules Active glycogen phosphorylase a Glucose 1-phosphate [Ca 2+ ] adenylyl cyclase [AMP] Glycogen Glycolysis Muscle contraction Glucose Blood glucose Myocyte H9251 FIGURE 15–26 Glycogen phosphorylase of liver as a glucose sensor. Glucose binding to an allosteric site of the phosphorylase a isozyme of liver induces a conformational change that exposes its phosphory- lated Ser residues to the action of phosphorylase a phosphatase 1(PP1). This phosphatase converts phosphorylase a to phosphorylase b, sharply reducing the activity of phosphorylase and slowing glycogen breakdown in response to high blood glucose. Insulin also acts indi- rectly to stimulate PP1 and slow glycogen breakdown. (active) CH 2 O P Allosteric sites empty 2 Glucose CH 2 O CH 2 O P phosphorylase a phosphatase (PP1) 2P i Glc CH 2 CH 2 OH OH (less active) Glc Glc Glc CH 2 OP P Insulin Phosphorylase a Phosphorylase a Phosphorylase b 8885d_c15_560-600 2/26/04 9:04 AM Page 585 mac76 mac76:385_reb: Glycogen Synthase Is Also Regulated by Phosphorylation and Dephosphorylation Like glycogen phosphorylase, glycogen synthase can ex- ist in phosphorylated and dephosphorylated forms (Fig. 15–27). Its active form, glycogen synthase a, is un- phosphorylated. Phosphorylation of the hydroxyl side chains of several Ser residues of both subunits converts glycogen synthase a to glycogen synthase b, which is inactive unless its allosteric activator, glucose 6- phosphate, is present. Glycogen synthase is remarkable for its ability to be phosphorylated on various residues by at least 11 different protein kinases. The most im- portant regulatory kinase is glycogen synthase kinase 3 (GSK3), which adds phosphoryl groups to three Ser residues near the carboxyl terminus of glycogen syn- thase, strongly inactivating it. The action of GSK3 is hi- erarchical; it cannot phosphorylate glycogen synthase until another protein kinase, casein kinase II (CKII), has first phosphorylated the glycogen synthase on a nearby residue, an event called priming (Fig. 15–28a). In liver, conversion of glycogen synthase b to the active form is promoted by PP1, which is bound to the glycogen particle. PP1 removes the phosphoryl groups from the three Ser residues phosphorylated by GSK3. Glucose 6-phosphate binds to an allosteric site on glyco- gen synthase b, making the enzyme a better substrate for dephosphorylation by PP1 and causing its activation. By analogy with glycogen phosphorylase, which acts as a glucose sensor, glycogen synthase can be regarded as a glucose 6-phosphate sensor. In muscle, a different phosphatase may have the role played by PP1 in liver, activating glycogen synthase by dephosphorylating it. Glycogen Synthase Kinase 3 Mediates the Actions of Insulin As we saw in Chapter 12, one way in which insulin trig- gers intracellular changes is by activating a protein ki- nase (protein kinase B, or PKB) that in turn phosphor- ylates and inactivates GSK3 (Fig. 15–29; see also Fig. 12–8). Phosphorylation of a Ser residue near the amino terminus of GSK3 converts that region of the protein to a pseudosubstrate, which folds into the site at which the priming phosphorylated Ser residue normally binds (Fig. 15–28b). This prevents GSK3 from binding the priming site of a real substrate, thereby inactivating the enzyme and tipping the balance in favor of dephosphor- ylation of glycogen synthase by PP1. Glycogen phos- phorylase can also affect the phosphorylation of glyco- gen synthase: active glycogen phosphorylase directly inhibits PP1, preventing it from activating glycogen syn- thase (Fig. 15–27). Although first discovered in its role in glycogen me- tabolism (hence the name glycogen synthase kinase), GSK3 clearly has a much broader role than the regula- tion of glycogen synthase. It mediates signaling by in- sulin and other growth factors and nutrients, and it acts in the specification of cell fates during embryonic de- velopment. Among its targets are cytoskeletal proteins Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen586 Insulin ADP ATP 3ADP 3ATP GSK3 CKII HO HO HO Glycogen synthase a Glycogen synthase b Inactive PP1 3P i Active GlucoseInsulin Glucose 6-phosphate Glucagon, epinephrine Phosphoserines near carboxyl terminus P P P FIGURE 15–27 Effects of GSK3 on glycogen synthase activity. Glycogen synthase a, the active form, has three Ser residues near its carboxyl terminus, which are phosphorylated by glycogen synthase kinase 3 (GSK3). This converts glycogen synthase to the inactive (b) form (GSb). GSK3 action requires prior phosphorylation (priming) by casein kinase (CKII). Insulin triggers activation of glycogen synthase b by blocking the activity of GSK3 (see the pathway for this action in Fig. 12–8) and activating a phosphoprotein phosphatase (PP1 in muscle, another phosphatase in liver). In muscle, epinephrine acti- vates PKA, which phosphorylates the glycogen-targeting protein GM (see Fig. 15–30) on a site that causes dissociation of PP1 from glycogen. Glucose 6-phosphate favors dephosphorylation of glycogen synthase by binding to it and promoting a conformation that is a good substrate for PP1. Glucose also promotes dephosphorylation; the binding of glucose to glycogen phosphorylase a forces a conformational change that favors dephosphorylation to glycogen phosphorylase b, thus re- lieving its inhibition of PP1 (see Fig. 15–29). 8885d_c15_586 2/26/04 2:02 PM Page 586 mac76 mac76:385_reb: 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown 587 FIGURE 15–28 Priming of GSK3 phosphorylation of glycogen syn- thase. (a) Glycogen synthase kinase 3 first associates with its substrate (glycogen synthase) by interaction between three positively charged residues (Arg 96 , Arg 180 , Lys 205 ) and a phosphoserine residue at posi- tion H110014 in the substrate. (For orientation, the Ser or Thr residue to be phosphorylated in the substrate is assigned the index 0. Residues on the amino-terminal side of this residue are numbered H110021, H110022, and so forth; residues on the carboxyl-terminal side are numbered H110011, H110012, and so forth.) This association aligns the active site of the enzyme with a Ser residue at position 0, which it phosphorylates. This creates a new priming site, and the enzyme moves down the protein to phosphory- late the Ser residue at position H110024, and then the Ser at H110028. (b) GSK3 has a Ser residue near its amino terminus that can be phosphorylated by PKA or PKB (see Fig. 15–29). This produces a “pseudosubstrate” region in GSK3 that folds into the priming site and makes the active site inaccessible to another protein substrate, inhibiting GSK3 until the priming phosphoryl group of its pseudosubstrate region is removed by PP1. Other proteins that are substrates for GSK3 also have a priming site at position H110014, which must be phosphorylated by another protein kinase before GSK3 can act on them. (a) GSK3 Arg 96 Arg 180 Lys 205 PP P PHH H ATP A SSS S S S SVLRQEEED +40–4–8 Glycogen synthase Active site Priming site phosphorylated by casein kinase II Ser residues phosphorylated in glycogen synthase O – O – O O P O H O H O H O GSK3 (b) RT Pseudosubstrate RPH 3 N + ST F E S C A +40 O – O – O O P Active Inactive 3P i PP1 Cytosol OH OH OH PKB P GSK3 GSK3 P P P PIP 3 PIP 2 PDK-1 Insulin Insulin receptor OH IRS-1 IRS-1 P PI-3K Plasma membrane Glycogen synthase b Glycogen synthase a Inactive Active FIGURE 15–29 The path from insulin to GSK3 and glycogen syn- thase. Insulin binding to its receptor activates a tyrosine protein ki- nase in the receptor, which phosphorylates insulin receptor substrate-1 (IRS-1). The phosphotyrosine in this protein is then bound by phos- phatidylinositol 3-kinase (PI-3K), which converts phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the membrane to phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). A protein kinase (PDK-1) that is activated when bound to PIP 3 activates a second protein kinase (PKB), which phosphorylates glycogen synthase kinase 3 (GSK3) in its pseudosub- strate region, inactivating it by the mechanisms shown in Figure 15–28b. The inactivation of GSK3 allows phosphoprotein phosphatase 1 (PP1) to dephosphorylate glycogen synthase, converting it to its ac- tive form. In this way, insulin stimulates glycogen synthesis. (See Fig. 12–8 for more details on insulin action.) 8885d_c15_587 2/26/04 2:02 PM Page 587 mac76 mac76:385_reb: and proteins essential for mRNA and protein synthesis. These targets, like glycogen synthase, must first un- dergo a priming phosphorylation by another protein ki- nase before they can be phosphorylated by GSK3. Phosphoprotein Phosphatase 1 Is Central to Glycogen Metabolism A single enzyme, PP1, can remove phosphoryl groups from all three of the enzymes phosphorylated in re- sponse to glucagon (liver) and epinephrine (liver and muscle): phosphorylase kinase, glycogen phosphory- lase, and glycogen synthase. Insulin stimulates glycogen synthesis by activating PP1 and by inactivating GSK3. PP1 does not exist free in the cytosol, but is tightly bound to its target proteins by one of a family of glycogen-targeting proteins that bind glycogen and each of the three enzymes, glycogen phosphorylase, phosphorylase kinase, and glycogen synthase (Fig. 15–30). PP1 is itself subject to covalent and allosteric regulation; it is inactivated when phosphorylated by PKA and is allosterically activated by glucose 6-phosphate. Transport into Cells Can Limit Glucose Utilization The passive uptake of glucose by muscle and adipose tissue is catalyzed by the GLUT4 transporter described in Box 11–2. In the absence of insulin, most GLUT4 mol- ecules are sequestered in membrane vesicles within the cell, but when blood glucose rises, release of insulin trig- gers GLUT4 movement to the plasma membrane. Glu- cose transport into hepatocytes involves a different, high-capacity transporter, GLUT2, which is always pres- ent in the plasma membrane. It catalyzes facilitated dif- fusion of glucose in both directions, at a rate high enough to ensure virtually instantaneous equilibration of glucose concentration in the blood and in the hepa- tocyte cytosol. In its role as a glucose sensor, the glyco- gen phosphorylase of hepatocytes is essentially meas- uring the glucose level in blood. Allosteric and Hormonal Signals Coordinate Carbohydrate Metabolism Having looked at the mechanisms that regulate individ- ual enzymes, we can now consider the overall shifts in carbohydrate metabolism that occur in the well-fed state, during fasting, and in the fight-or-flight re- sponse—signaled by insulin, glucagon, and epinephrine, respectively. We need to contrast two cases in which regulation serves different ends: (1) the role of hepato- cytes in supplying glucose to the blood, and (2) the self- ish use of carbohydrate fuels by nonhepatic tissues, typ- ified by skeletal muscle (the myocyte), to support their own activities. After ingestion of a carbohydrate-rich meal, the elevation of blood glucose triggers insulin release (Fig. 15–31, top). In a hepatocyte, insulin has two immediate effects: it inactivates GSK3, acting through the cascade shown in Figure 15–29, and activates a protein phos- phatase, perhaps PP1. These two actions fully activate glycogen synthase. PP1 also inactivates glycogen phos- phorylase a and phosphorylase kinase by dephosphory- lating both, effectively stopping glycogen breakdown. Glu- cose enters the hepatocyte through the high-capacity transporter GLUT2, always present in the plasma mem- brane, and the elevated intracellular glucose leads to dis- sociation of hexokinase IV (glucokinase) from its nuclear regulator protein. Hexokinase IV enters the cytosol and phosphorylates glucose, stimulating glycolysis and sup- plying the precursor for glycogen synthesis. Under these conditions, hepatocytes use the excess glucose in the blood to synthesize glycogen, up to the limit of about 10% of the total weight of the liver. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen588 Phosphorylase kinase GM Glycogen phosphorylase Glycogen synthase PKA insulin- sensitive kinase Inhibitor 1 epinephrine Glycogen granule Phosphorylated inhibitor 1 binds and inactivates PP1 insulin 1 2 GM PP1 PP1 P GM P P P P FIGURE 15–30 Glycogen-targeting protein GM. The glycogen-targeting protein GM is one of a family of proteins that bind other proteins (including PP1) to glycogen particles. GM can be phosphorylated in two different positions in response to insulin or epinephrine. 1 Insulin-stimulated phosphorylation of GM site 1 activates PP1, which dephosphorylates phosphorylase kinase, glycogen phosphorylase, and glycogen synthase. 2 Epinephrine- stimulated phosphorylation of GM site 2 causes dissociation of PP1 from the glycogen particle, preventing its access to glycogen phosphorylase and glycogen synthase. PKA also phosphorylates a protein (inhibitor 1) that, when phosphorylated, inhibits PP1. By these means, insulin inhibits glycogen breakdown and stimulates glycogen synthesis, and epinephrine (or glucagon in the liver) has the opposite effects. 8885d_c15_560-600 2/26/04 9:04 AM Page 588 mac76 mac76:385_reb: liver produces glucose 6-phosphate by glycogen break- down and by gluconeogenesis, and it stops using glucose to fuel glycolysis or make glycogen, maximizing the amount of glucose it can release to the blood. This re- lease of glucose is possible only in liver, because other tissues lack glucose 6-phosphatase (Fig. 15–6). The physiology of skeletal muscle differs from that of liver in three ways important to our discussion of metabolic regulation (Fig. 15–32): (1) muscle uses its stored glycogen only for its own needs; (2) as it goes from rest to vigorous contraction, muscle undergoes very large changes in its demand for ATP, which is supported by glycolysis; (3) muscle lacks the enzymatic machin- ery for gluconeogenesis. The regulation of carbohydrate 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown 589 Between meals, or during an extended fast, the drop in blood glucose triggers the release of glucagon, which, acting through the cascade shown in Figure 15–25, ac- tivates PKA. PKA mediates all the effects of glucagon (Fig. 15–31, bottom). It phosphorylates phosphorylase kinase, activating it and leading to the activation of glyco- gen phosphorylase. It phosphorylates glycogen synthase, inactivating it and blocking glycogen synthesis. It phos- phorylates PFK-2/FBPase-2, leading to a drop in the con- centration of the regulator fructose 2,6-bisphosphate, which has the effect of inactivating the glycolytic enzyme PFK-1 and activating the gluconeogenic enzyme FBPase- 1. And it phosphorylates and inactivates the glycolytic enzyme pyruvate kinase. Under these conditions, the FIGURE 15–31 Regulation of carbohydrate metabolism in the hepatocyte. Arrows indicate causal relationships between the changes they connect. gA nhB means that a decrease in A causes an increase in B. Pink arrows connect events that result from high blood glucose; blue arrows connect events that result from low blood glucose. High blood glucose Insulin Insulin-sensitive protein kinase Phosphorylase kinase PKB GSK-3PP1 Glycogen phosphorylase Glycogen breakdown Glycogen synthesis Glycolysis Glycogen breakdown Glycogen synthesis Glycogen phosphorylase Phosphorylase kinase FBPase-2 PFK-2 PKA cAMP Glucagon Low blood glucose Pyruvate kinase L Glycogen synthase PFK-1 F26BP Glycogen synthase Synthesis of hexokinase II, PFK-1, pyruvate kinase GLUT2 [Glucose] inside Glycolysis 8885d_c15_589 2/26/04 2:02 PM Page 589 mac76 mac76:385_reb: Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen590 metabolism in muscle reflects these differences from liver. First, myocytes lack receptors for glucagon. Sec- ond, the muscle isozyme of pyruvate kinase is not phos- phorylated by PKA, so glycolysis is not turned off when [cAMP] is high. In fact, cAMP increases the rate of gly- colysis in muscle, probably by activating glycogen phos- phorylase. When epinephrine is released into the blood in a fight-or-flight situation, PKA is activated by the rise in [cAMP], and phosphorylates and activates glycogen phosphorylase kinase. The resulting phosphorylation and activation of glycogen phosphorylase results in faster glycogen breakdown. Epinephrine is not released under low-stress conditions, but with each neuronal stimulation of muscle contraction, cytosolic [Ca 2H11001 ] rises briefly and activates phosphorylase kinase through its calmodulin subunit. Elevated insulin triggers increased glycogen syn- thesis in myocytes by activating PP1 and inactivating GSK3. Unlike hepatocytes, myocytes have a reserve of GLUT4 sequestered in intracellular vesicles. Insulin trig- gers their movement to the plasma membrane, where they allow increased glucose uptake. In response to in- sulin, therefore, myocytes help to lower blood glucose by increasing their rates of glucose uptake, glycogen synthesis, and glycolysis. Insulin Changes the Expression of Many Genes Involved in Carbohydrate and Fat Metabolism In addition to its effects on the activity of existing en- zymes, insulin also regulates the expression of as many as 150 genes, including some related to fuel metabolism (Fig. 15–31; Table 15–3). Insulin stimulates the tran- scription of the genes that encode hexokinases II and IV, PFK-1, pyruvate kinase, and the bifunctional enzyme PFK-2/FBPase-2 (all involved in glycolysis and its regu- lation), several enzymes involved in fatty acid synthesis, and two enzymes that generate the reductant for fatty acid synthesis (NADPH) via the pentose phosphate pathway (glucose 6-phosphate dehydrogenase and 6- phosphogluconate dehydrogenase). Insulin also slows the expression of the genes for two enzymes of gluconeoge- nesis (PEP carboxykinase and glucose 6-phosphatase). These effects take place on a longer time scale (minutes to hours) than those mediated by covalent alteration of enzymes, but the impact on metabolism can be very sig- nificant. When the diet provides an excess of glucose, the resulting rise in insulin increases the synthesis of glucose- metabolizing proteins, and glucose becomes the fuel of choice (via glycolysis) for liver, adipose tissue, and mus- cle. In liver and adipose tissue, glucose is converted to glycogen and triacylglycerols for temporary storage. Carbohydrate and Lipid Metabolism Are Integrated by Hormonal and Allosteric Mechanisms As complex as the regulation of carbohydrate metabo- lism is, it is far from the whole story of fuel metabolism. The metabolism of fats and fatty acids is very closely tied to that of carbohydrates. Hormonal signals such as insulin and changes in diet or exercise are equally important in regulating fat metabolism and integrating it with that of carbohydrates. We shall return to this overall metabolic integration in mammals in Chapter 23, Change in gene expression Pathway Increased expression Hexokinase II Glycolysis Hexokinase IV Glycolysis Phosphofructokinase-1 (PFK-1) Glycolysis Pyruvate kinase Glycolysis PFK-2/FBPase-2 Regulation of glycolysis/gluconeogenesis Glucose 6-phosphate dehydrogenase Pentose phosphate pathway (NADPH) 6-Phosphogluconate dehydrogenase Pentose phosphate pathway (NADPH) Pyruvate dehydrogenase Fatty acid synthesis Acetyl-CoA carboxylase Fatty acid synthesis Malic enzyme Fatty acid synthesis (NADPH) ATP-citrate lyase Fatty acid synthesis (provides acetyl-CoA) Fatty acid synthase complex Fatty acid synthesis Stearoyl-CoA dehydrogenase Fatty acid desaturation Acyl-CoA–glycerol transferases Triacylglycerol synthesis Decreased expression PEP carboxykinase Gluconeogenesis Glucose 6-phosphatase (catalytic subunit) Glucose release to blood TABLE 15–3 Some of the Genes Regulated by Insulin 8885d_c15_560-600 2/26/04 9:04 AM Page 590 mac76 mac76:385_reb: after first considering the metabolic pathways for fats and amino acids (Chapters 17 and 18). The message we wish to convey here is that metabolic pathways are over- laid with complex regulatory controls that are exquis- itely sensitive to changes in metabolic circumstances. These mechanisms act to adjust the flow of metabolites through various metabolic pathways as needed by the cell and organism, and do so without causing major changes in the concentrations of intermediates shared with other pathways. SUMMARY 15.4 Coordinated Regulation of Glycogen Synthesis and Breakdown ■ Glycogen phosphorylase is activated in response to glucagon or epinephrine, which raise [cAMP] and activate PKA. PKA phosphorylates and activates phosphorylase kinase, which converts glycogen phosphorylase b to its active a form. Phosphoprotein phosphatase 1 (PP1) reverses the phosphorylation of glycogen phosphorylase a, inactivating it. Glucose binds to the liver isozyme of glycogen phosphorylase a, favoring its dephosphorylation and inactivation. ■ Glycogen synthase a is inactivated by phosphorylation catalyzed by GSK3. Insulin blocks GSK3. PP1, which is activated by insulin, reverses the inhibition by dephosphorylating glycogen synthase b. ■ Insulin increases glucose uptake into myocytes and adipocytes by triggering movement of the glucose transporter GLUT4 to the plasma membrane. ■ Insulin stimulates the synthesis of hexokinases II and IV, PFK-1, pyruvate kinase, and several enzymes involved in lipid synthesis. Insulin stimulates glycogen synthesis in muscle and liver. ■ In liver, glucagon stimulates glycogen breakdown and gluconeogenesis while blocking glycolysis, thereby sparing glucose for export to the brain and other tissues. ■ In muscle, epinephrine stimulates glycogen breakdown and glycolysis, providing ATP to support contraction. 15.5 Analysis of Metabolic Control For every complex problem there is a simple solution. And it is always wrong. —H. L. Mencken, A Mencken Chrestomathy, 1949 Beginning with Eduard Buchner’s discovery (c. 1900) that an extract of broken yeast cells could convert glu- cose to ethanol and CO 2 , a major thrust of biochemical research was to deduce the steps by which this trans- formation occurred and to purify and characterize the enzymes that catalyzed each step. By the middle of the twentieth century, all ten en- zymes of the glycolytic path- way had been purified and characterized. In the next 50 years much was learned about the regulation of these en- zymes by intracellular and ex- tracellular signals, through the kinds of allosteric and cova- lent mechanisms we have de- scribed in this chapter. The conventional wisdom was that in a linear pathway such as glycolysis, catalysis by one enzyme must be the slowest and must therefore determine the rate of metabolite flow, or flux, through the whole pathway. For glycoly- sis, PFK-1 was considered the rate-limiting enzyme, be- cause it was known to be closely regulated by fructose 2,6-bisphosphate and other allosteric effectors. 15.5 Analysis of Metabolic Control 591 Epinephrine Glucagon Liver Muscle Glycogen Glycogen Glycogenolysis Glucose 6-phosphate Glucose 6-phosphate Blood glucose Glycolysis Gluconeogenesis Pyruvate Pyruvate FIGURE 15–32 Difference in the regulation of carbohydrate metabolism in liver and muscle. In liver, either glucagon (indicating low blood glucose) or epinephrine (signaling the need to fight or flee) has the effect of maximizing the output of glucose into the bloodstream. In muscle, epinephrine increases glycogen breakdown and glycolysis, which together provide fuel to produce the ATP needed for muscle contraction. Eduard Buchner, 1860–1917 8885d_c15_560-600 2/26/04 9:04 AM Page 591 mac76 mac76:385_reb: With the advent of genetic engineering technology, it became possible to test this “single rate-determining step” hypothesis by increasing the concentration of the enzyme that catalyzes the “rate-limiting step” in a path- way and determining whether flux through the pathway increases proportionally. More often than not, it does not do so: the simple solution (a single rate-determining step) is wrong. It has now become clear that in most pathways the control of flux is distributed among sev- eral enzymes, and the extent to which each contributes to the control varies with metabolic circumstances—the supply of the starting material (say, glucose), the sup- ply of oxygen, the need for other products derived from intermediates of the pathway (say, glucose 6-phosphate for the pentose phosphate pathway in cells synthesiz- ing large amounts of nucleotides), the effects of metabo- lites with regulatory roles, and the hormonal status of the organism (the levels of insulin and glucagon), among other factors. Why are we interested in what limits the flux through a pathway? To understand the action of hor- mones or drugs, or the pathology that results from a fail- ure of metabolic regulation, we must know where con- trol is exercised. If researchers wish to develop a drug that stimulates or inhibits a pathway, the logical target is the enzyme that has the greatest impact on the flux through that pathway. And the bioengineering of a mi- croorganism to overproduce a product of commercial value (p. 315) requires a knowledge of what limits the flux of metabolites toward that product. The Contribution of Each Enzyme to Flux through a Pathway Is Experimentally Measurable There are several ways to determine experimentally how a change in the activity of one enzyme in a path- way affects metabolite flux through that pathway. Con- sider the experimental results shown in Figure 15–33. When a sample of rat liver was homogenized to re- lease all soluble enzymes, the extract carried out the gly- colytic conversion of glucose to fructose 1,6- bispho- sphate at a measurable rate. (This experiment, for simplicity, focused on just the first part of the gly- colytic pathway.) When increasing amounts of purified hexokinase IV were added to the extract, the rate of gly- colysis progressively increased. The addition of purified PFK-1 to the extract also increased the rate of glycoly- sis, but not as dramatically as did hexokinase. Purified phosphohexose isomerase was without effect. These re- sults suggest that hexokinase and PFK-1 both contri- bute to setting the flux through the pathway (hexokinase more than PFK-1), and that phosphohexose isomerase does not. Similar experiments can be done on intact cells or organisms, using specific inhibitors or activators to change the activity of one enzyme while observing the effect on flux through the pathway. The amount of an enzyme can also be altered genetically; bioengineering can produce a cell that makes extra copies of the en- zyme under investigation or has a version of the enzyme that is less active than the normal enzyme. Increasing the concentration of an enzyme genetically sometimes has significant effects on flux; sometimes it has no effect. Three critical parameters, which together describe the responsiveness of a pathway to changes in meta- bolic circumstances, lie at the center of metabolic con- trol analysis. We turn now to a qualitative description of these parameters and their meaning in the context of a living cell. In Box 15–3 we will provide a more rig- orous quantitative discussion. The Control Coefficient Quantifies the Effect of a Change in Enzyme Activity on Metabolite Flux through a Pathway Quantitative data obtained as described in Figure 15–33 can be used to calculate a flux control coefficient, C, for each enzyme in a pathway. This coefficient ex- presses the relative contribution of each enzyme to set- ting the rate at which metabolites flow through the pathway—that is, the flux, J. C can have any value from 0.0 (for an enzyme with no impact on the flux) to 1.0 (for an enzyme that wholly determines the flux). An en- zyme can also have a negative flux control coefficient. In a branched pathway, an enzyme in one branch, by drawing intermediates away from the other branch, can have a negative impact on the flux through that other branch (Fig. 15–34). C is not a constant, and it is not Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen592 Glycolytic flux 0.10 0.08 0.06 0.04 0.02 0.00 0 0.5 1.0 1.5 Enzyme added (arbitrary units) 2.0 2.5 3.0 Hexokinase IV Phosphofructokinase-1 Phosphohexose isomerase FIGURE 15–33 Dependence of glycolytic flux in a rat liver homog- enate on added enzymes. Purified enzymes in the amounts shown on the x axis were added to an extract of liver carrying out glycolysis in vitro. The increase in flux through the pathway is shown on the y axis. 8885d_c15_592 2/26/04 2:03 PM Page 592 mac76 mac76:385_reb: intrinsic to a single enzyme; it is a function of the whole system of enzymes, and its value depends on the con- centrations of substrates and effectors. When real data from the experiment on glycolysis in a rat liver extract (Fig. 15–33) were subjected to this kind of analysis, investigators found flux control coeffi- cients (for enzymes at the concentrations found in the extract) of 0.79 for hexokinase, 0.21 for PFK-1, and 0.0 for phosphohexose isomerase. It is not just fortuitous that these values add up to 1.0; we can show that for any complete pathway, the sum of the flux control co- efficients must equal unity. The Elasticity Coefficient Is Related to an Enzyme’s Responsiveness to Changes in Metabolite or Regulator Concentrations A second parameter, the elasticity coefficient, H9255, ex- presses quantitatively the responsiveness of a single en- zyme to changes in the concentration of a metabolite or regulator; it is a function of the enzyme’s intrinsic ki- netic properties. For example, an enzyme with typical Michaelis-Menten kinetics shows a hyperbolic response to increasing substrate concentration (Fig. 15–35). At low concentrations of substrate (say, 0.1 K m ) each in- crement in substrate concentration results in a compa- rable increase in enzymatic activity, yielding an H9255 near 1.0. At relatively high substrate concentrations (say, 10 K m ), increasing the substrate concentration has little ef- fect on the reaction rate, because the enzyme is already saturated with substrate. The elasticity in this case ap- proaches zero. For allosteric enzymes that show posi- tive cooperativity, H9255 may exceed 1.0, but it cannot ex- ceed the Hill coefficient. Recall that the Hill coefficient is a measure of the degree of cooperativity, typically be- tween 1.0 and 4.0 (p. 167). The Response Coefficient Expresses the Effect of an Outside Controller on Flux through a Pathway We can also derive a quantitative expression for the rel- ative impact of an outside factor (such as a hormone or growth factor), which is neither a metabolite nor an en- zyme in the pathway, on the flux through the pathway. The experiment would measure the flux through the pathway (glycolysis, in this case) at various levels of the parameter P (the insulin concentration, for example) to obtain the response coefficient, R, which expresses the change in pathway flux when P ([insulin]) changes. The three coefficients C, H9255, and R are related in a simple way: the responsiveness (R) of a pathway to an outside factor that affects a certain enzyme is a func- tion of (1) how sensitive the pathway is to changes in the activity of that enzyme (the control coefficient, C) and (2) how sensitive that specific enzyme is to changes in the outside factor (the elasticity, H9255): R H11005 C H11554 H9255 Each enzyme in the pathway can be examined in this way, and the effects of any of several outside factors on flux through the pathway can be separately determined. Thus, in principle, we can predict how the flux of sub- strate through a series of enzymatic steps will change when there is a change in one or more controlling fac- tors external to the pathway. Box 15–3 shows how these qualitative concepts are treated quantitatively. Metabolic Control Analysis Has Been Applied to Carbohydrate Metabolism, with Surprising Results Metabolic control analysis provides a framework within which we can think quantitatively about regulation, in- terpret the significance of the regulatory properties of each enzyme in a pathway, identify the steps that most affect the flux through the pathway, and distinguish between regulatory mechanisms that act to maintain metabolite concentrations and control mechanisms that actually alter the flux through the pathway. Analy- sis of the glycolytic pathway in yeast, for example, has 15.5 Analysis of Metabolic Control 593 V max v K m [S] H9280 ≈ 0.0 H9280 ≈ 1.0 A B C D E C 4 H11005 H110020.2 C 1 H11005 0.3 C 2 H11005 0.0 C 3 H11005 0.9 FIGURE 15–34 Flux control coefficient, C, in a branched metabolic pathway. In this simple pathway, the intermediate B has two alterna- tive fates. To the extent that reaction B n E draws B away from the pathway A n D, it controls that pathway, which will result in a neg- ative flux control coefficient for the enzyme that catalyzes step B n E. Note that the sum of all four coefficients equals 1.0, as it must. FIGURE 15–35 Elasticity coefficient, H9255, of an enzyme with typical Michaelis-Menten kinetics. At substrate concentrations far below the K m , each increase in [S] produces a correspondingly large increase in the reaction velocity, v. For this region of the curve, the enzyme has an elasticity, H9255, of about 1.0. At [S] >> K m , increasing [S] has little ef- fect on v; H9255 here is close to 0.0. 8885d_c15_560-600 2/26/04 9:04 AM Page 593 mac76 mac76:385_reb: revealed an unexpectedly low flux control coefficient for PFK-1, which, because of its known elaborate allosteric regulation, has been viewed as the main point of flux control—the “rate-determining step”—in glycolysis. Experimentally raising the level of PFK-1 fivefold led to a change in flux through glycolysis of less than 10%, suggesting that the real role of PFK-1 regulation is not to control flux through glycolysis but to mediate metabolite homeostasis—to prevent large changes in metabolite concentrations when the flux through gly- colysis increases in response to elevated blood glucose or insulin. Recall that the study of glycolysis in a liver Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen594 BOX 15–3 WORKING IN BIOCHEMISTRY Metabolic Control Analysis: Quantitative Aspects The factors that influence the flow of intermediates (flux) through a pathway may be determined quanti- tatively by experiment and expressed in terms useful for predicting the change in flux when some factor in- volved in the pathway changes. Consider the simple reaction sequence in Figure 1, in which a substrate X (say, glucose) is converted in several steps to a prod- uct Z (perhaps pyruvate, formed glycolytically). A later enzyme in the pathway is a dehydrogenase (ydh) that acts on substrate Y. Because the action of a de- hydrogenase is easily measured (see Fig. 13–15), we can use the flux (J) through this step (J ydh ) to meas- ure the flux through the whole path. We manipulate experimentally the level of an early enzyme in the pathway (xase, which acts on the substrate X) and measure the flux through the path (J ydh ) for several levels of the enzyme xase. the ratio H11128J ydh /H11128E xase . However, its usefulness is lim- ited because its value depends on the units used to express flux and enzyme activity. By expressing the fractional changes in flux and enzyme activity, H11128J ydh /J ydh , and H11128E xase /E xase , we obtain a unitless ex- pression for the flux control coefficient, C J ydh xase : C Jydh xase ≈ / (1) This can be rearranged to C Jydh xase ≈ H11554 which is mathematically identical to C Jydh xase H11005 This equation suggests a simple graphical means for determining the flux control coefficient: C J ydh xase is the slope of the tangent to the plot of ln J ydh versus ln E xase , which can be obtained by replotting the exper- imental data in Figure 2a to obtain Figure 2b. Notice that C J ydh xase is not a constant; it depends on the start- ing E xase from which the change in enzyme level takes place. For the cases shown in Figure 2, C J ydh xase is about 1.0 at the lowest E xase , but only about 0.2 at high E xase . A value near 1.0 for C J ydh xase means that the enzyme’s concentration wholly determines the flux through the pathway; a value near 0.0 means that the enzyme’s concentration does not limit the flux through the path. Unless the flux control coefficient is greater than about 0.5, changes in the activity of the enzyme will not have a strong effect on the flux. The elasticity, H9255, of an enzyme is a measure of how that enzyme’s catalytic activity changes when the concentration of a metabolite—substrate, product, or effector—changes. It is obtained from an experimen- tal plot of the rate of the reaction catalyzed by the en- zyme versus the concentration of the metabolite, at metabolite concentrations that prevail in the cell. By arguments analogous to those used to derive C, we can show H9255 to be the slope of the tangent to a plot of H11128ln J ydh H5007H5007 H11128ln Exase Exase H5007 Jydh H11128Jydh H5007 H11128Exase H11128E xase H5007 Exase H11128J ydh H5007 Jydh FIGURE 1 Flux through a hypothetical multienzyme pathway. X xase ydh J xase J ydh S 1 S 6 multistep Y Z multistep The relationship between the flux through the pathway from X to Z in the intact cell and the con- centration of each enzyme in the path should be hy- perbolic, with virtually no flux at infinitely low enzyme and near-maximum flux at very high enzyme activity. In a plot of J ydh against the concentration of xase, E xase , the change of flux with a small change of enzyme is H11128J ydh /H11128E xase , which is simply the slope of the tangent to the curve at any concentration of enzyme, E xase , and which tends toward zero at saturating E xase . At low E xase , the slope is steep; the flux increases with each incremental increase in enzyme activity. At very high E xase , the slope is much smaller; the system is less re- sponsive to added xase, because it is already present in excess over the other enzymes in the pathway. To show quantitatively the dependence of flux through the pathway, H11128J ydh , on H11128E xase , we could use 8885d_c15_594 2/26/04 2:03 PM Page 594 mac76 mac76:385_reb: extract (Fig. 15–33) also yielded a flux control coeffi- cient that contradicted the conventional wisdom; it showed that hexokinase, not PFK-1, is most influential in setting the flux through glycolysis. We must note here that a liver extract is far from equivalent to a hepato- cyte; the ideal way to study flux control is by manipu- lating one enzyme at a time in the living cell. This is already feasible in some cases. Investigators have used nuclear magnetic resonance (NMR) as a noninvasive means to determine the concen- tration of glycogen and metabolites in the five-step path- way from glucose in the blood to glycogen in myocytes 15.5 Analysis of Metabolic Control 595 ln V versus ln [substrate, or product, or effector]: H9255 xase S H11005 H11554 H11005 For an enzyme with typical Michaelis-Menten kinet- ics, the value of H9255 ranges from about 1 at substrate concentrations far below K m to near 0 as V max is ap- proached. Allosteric enzymes can have elasticities greater than 1.0, but not larger than their Hill coeffi- cients (p. 167). Finally, the effect of controllers outside the path- way itself (that is, not metabolites) can be measured and expressed as the response coefficient, R. The change in flux through the pathway is measured for changes in the concentration of the controlling pa- rameter P, and R is defined in a form analogous to that H11128ln ?V xase ? H5007H5007 H11128ln S S H5007 V xase H11128V xase H5007 H11128S of Equation 1, yielding the expression R Jydh P H11005 H11554 Using the same logic and graphical methods as de- scribed above for determining C, we can obtain R as the slope of the tangent to the plot of ln J versus ln P. The three coefficients we have described are re- lated in this simple way: R Jydh P H11005 C Jydh xase H11554 H9255 xase P Thus the responsiveness of each enzyme in a pathway to a change in an outside controlling factor is a sim- ple function of two things: the control coefficient, a variable that expresses the extent to which that en- zyme influences the flux under a given set of condi- tions, and the elasticity, an intrinsic property of the enzyme that reflects its sensitivity to substrate and effector concentrations. P H5007 J ydh H11128J ydh H5007 H11128P FIGURE 2 The flux control coefficient. (a) Typical variation of the pathway flux, J ydh , measured at the step catalyzed by the enzyme ydh, as a function of the amount of the enzyme xase, E xase , which cat- alyzes an earlier step in the pathway. The flux control coefficient at (e,j) is the slope of the product of the tangent to the curve, H11128J ydh /H11128E xase , and the ratio (scaling factor), e/j. (b) On a double-logarithmic plot of the same curve, the flux control coefficient is the slope of the tangent to the curve. Flux, J ydh e j ?J ydh ?E xase ln J ydh ln e ln j ?ln J ydh ?ln E xase H11005 C J ydh xase (a) (b) Concentration of enzyme, E xase ln E xase 8885d_c15_595 2/26/04 2:03 PM Page 595 mac76 mac76:385_reb: (Fig. 15–36) in rat and human muscle. They found that the flux control coefficient for glycogen synthase was smaller than that for the steps catalyzed by the glucose transporter and hexokinase. This finding, too, contra- dicts the conventional wisdom that glycogen synthase is the locus of flux control and suggests that the im- portance of the phosphorylation/dephosphorylation of glycogen synthase is related instead to the maintenance of metabolite homeostasis—that is, regulation, not control. Two metabolites in this pathway, glucose and glucose 6-phosphate, are key intermediates in other pathways, including glycolysis, the pentose phosphate pathway, and the synthesis of glucosamine. Metabolic control analysis suggests that when the blood glucose level rises, insulin acts in muscle to (1) increase glucose transport into cells by bringing GLUT4 to the plasma membrane, (2) induce the synthesis of hexokinase, and (3) activate glycogen synthase by covalent alteration (Fig. 15–29). The first two effects of insulin increase glucose flux through the pathway (control), and the third serves to adapt the activity of glycogen synthase so that metabolite levels (glucose 6-phosphate, for ex- ample) will not change dramatically with the increased flux (regulation). Metabolic Control Analysis Suggests a General Method for Increasing Flux through a Pathway How could an investigator engineer a cell to increase the flux through one pathway without altering the concen- trations of other metabolites or the fluxes through other pathways? More than two decades ago Henrik Kacser predicted, on the basis of metabolic control analysis, that this could be accomplished by increasing the con- centrations of every enzyme in a pathway. The predic- tion has been confirmed in several experimental tests, and it also fits with the way cells normally control fluxes through a pathway. For example, when rats are fed a high-protein diet, they dispose of excess amino groups by converting them to urea in the urea cycle (Chapter 18). After such a dietary shift, the urea output increases fourfold, and the amount of all eight enzymes in the urea cycle increases two- to threefold. Similarly, when in- creased fatty acid oxidation is triggered by activation of the enzyme peroxisome proliferator-activated receptor H9253 (PPARH9253; see Fig. 21–22), synthesis of the whole set of oxidative enzymes is increased. With the growing use of DNA microarrays to study the expression of whole sets of genes in response to various perturbations, we should soon learn whether this is the general mecha- nism by which cells make long-term adjustments in the fluxes through specific pathways. SUMMARY 15.5 Analysis of Metabolic Control ■ Metabolic control analysis shows that control of the rate of metabolite flux through a pathway is distributed among several of the enzymes in that path. ■ The flux control coefficient, C, is an experimentally determined measure of the effect of an enzyme’s concentration on flux through a multienzyme pathway. It is characteristic of the whole system, not intrinsic to the enzyme. ■ The elasticity coefficient, H9255, of an enzyme is an experimentally determined measure of how responsive the enzyme is to changes in the concentration of a metabolite or regulator molecule. ■ The response coefficient, R, is the expression for the experimentally determined change in flux through a pathway in response to a regulatory hormone or second messenger. It is a function of C and H9255: R H11005 C H11554 H9255. ■ Some regulated enzymes control the flux through a pathway, while others rebalance the level of metabolites in response to the change in flux. This latter, rebalancing activity is regulation; the former activity is control. ■ Metabolic control analysis predicts that flux toward a desired product is most effectively increased by raising the concentration of all enzymes in the pathway. Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen596 Plasma membrane Capillary Glucose Insulin UDP-glucose glycogen synthase Glucose 1-phosphate Glucose 6-phosphate hexokinase Myocyte GLUT4 Glucose Glycogen FIGURE 15–36 Control of glycogen synthesis from blood glucose in myocytes. Insulin affects three of the five steps in this pathway, but it is the effects on transport and hexokinase activity, not the change in glycogen synthase activity, that increase the flux toward glycogen. 8885d_c15_560-600 2/26/04 9:04 AM Page 596 mac76 mac76:385_reb: Chapter 15 Further Reading 597 Key Terms glycogenolysis 562 glycolysis 562 gluconeogenesis 562 glycogenesis 562 debranching enzyme 562 sugar nucleotides 565 glycogenin 569 homeostasis 571 adenylate kinase 571 mass action ratio, Q 572 metabolic regulation 575 metabolic control 575 futile cycle 576 substrate cycle 576 GLUT 578 glucagon 581 fructose 2,6-bisphosphate 581 glycogen phosphorylase a 583 glycogen phosphorylase b 584 enzyme cascade 584 phosphoprotein phosphatase 1 (PP1) 584 glycogen synthase a 586 glycogen synthase b 586 glycogen synthase kinase 3 (GSK3) 586 priming 586 glycogen-targeting proteins 588 flux control coefficient, C 592 flux, J 592 elasticity coefficient, H9255 593 response coefficient, R 593 Terms in bold are defined in the glossary. Further Reading General and Historical Gibson, D.M. & Harris, R.A. (2002) Metabolic Regulation in Mammals, Taylor and Francis, New York. An excellent introduction to the regulation of metabolism in each of the major organs. Kornberg, A. (2001) Remembering our teachers. J. Biol. Chem. 276, 3–11. An appreciative description of the Coris’ laboratories and coworkers. Ochs, R.S., Hanson, R.W., & Hall, J. (eds) (1985) Metabolic Regulation, Elsevier Science Publishing Co. Inc., New York. A collection of short essays first published in Trends in Biochemical Sciences, better known as TIBS. Simoni, R.D., Hill, R.L., & Vaughan, M. (2002) Carbohydrate metabolism: glycogen phosphorylase and the work of Carl F. and Gerty T. Cori. J. Biol. Chem. 277 (www.jbc.org/cgi/content/ full/277/29/e18). A brief historical note with references to five classic papers by the Coris (online journal only). Metabolism of Glycogen in Animals Gibbons, B.J., Roach, P.J., & Hurley, T.D. (2002) Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin. J. Mol. Biol. 319, 463–477. Melendez-Hevia, E., Waddell, T.G., & Shelton, E.D. (1993) Optimization of molecular design in the evolution of metabolism: the glycogen molecule. Biochem. J. 295, 477–483. Comparison of theoretical and experimental aspects of glycogen structure. Regulation of Metabolic Pathways Barford, D. (1999) Structural studies of reversible protein phosphorylation and protein phosphatases. Biochem. Soc. Trans. 27, 751–766. An intermediate-level review. Coordinated Regulation of Glycolysis and Gluconeogenesis de la Iglesia, N., Mukhtar, M., Seoane, J., Guinovart, J.J., & Agius, L. (2000) The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J. Biol. Chem. 275, 10,597–10,603. Report of the experimental determination of the flux control coefficients for glucokinase and the glucokinase regulatory protein in hepatocytes. Hue, L. & Rider, M.H. (1987) Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem. J. 245, 313–324. Nordlie, R.C., Foster, J.D., & Lange, A.J. (1999) Regulation of glucose production by the liver. Annu. Rev. Nutr. 19, 379–406. Advanced review. Okar, D.A., Manzano, A., Navarro-Sabate, A., Riera, L., Bartrons, R., & Lange, A.J. (2001) PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem. Sci. 26, 30–35. A brief review of the bifunctional kinase/phosphatase. Pilkis, S.J. & Granner, D.K. (1992) Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54, 885–909. Schirmer, T. & Evans, P.R. (1990) Structural basis of the allosteric behavior of phosphofructokinase. Nature 343, 140–145. van Shaftingen, E. & Gerin, I. (2002) The glucose-6- phosphatase system. Biochem. J. 362, 513–532. Veech, R.L. (2003) A humble hexose monophosphate pathway metabolite regulates short- and long-term control of lipogenesis. Proc. Natl. Acad. Sci. USA 100, 5578–5580. Short review of the work from K. Uyeda’s laboratory on the role of xylulose 5-phosphate in carbohydrate and fat metabolism; Uyeda’s papers are cited here. Yamada, K. & Noguchi, T. (1999) Nutrient and hormonal regula- tion of pyruvate kinase gene expression. Biochem. J. 337, 1–11. Detailed review of recent work on the genes and proteins of this system and their regulation. Coordinated Regulation of Glycogen Synthesis and Breakdown Barford, D., Hu, S.-H., & Johnson, L.N. (1991) Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP. J. Mol. Biol. 218, 233–260. 8885d_c15_560-600 2/26/04 9:04 AM Page 597 mac76 mac76:385_reb: Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen598 Clear discussion of the regulatory changes in the structure of glycogen phosphorylase, based on the structures (from x-ray diffraction studies) of the active and less active forms of the enzyme. Frame, S. & Cohen, P. (2001) GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359, 1–16. Review of the roles of GSK3 in carbohydrate metabolism and in other regulatory phenomena. Harwood, A.J. (2001) Regulation of GSK-3: a cellular multiprocessor. Cell 105, 821–824. Short review of several regulatory roles of GSK3. Hudson, J.W., Golding, G.B., & Crerar, M.M. (1993) Evolution of allosteric control in glycogen phosphorylase. J. Mol. Biol. 234, 700–721. Newgard, C.B., Brady, M.J., O’Doherty, R.M., & Saltiel, A.R. (2000) Organizing glucose disposal: emerging roles of the glycogen targeting subunits of protein phosphatase-1. Diabetes 49, 1967–1977. Intermediate-level review. Radziuk, J. & Pye, S. (2001) Hepatic glucose uptake, gluconeogenesis and the regulation of glycogen synthesis. Diabetes/Metab. Res. Rev. 17, 250–272. Advanced review. Stalmans, W., Keppens, S., & Bollen, M. (1998) Specific features of glycogen metabolism in the liver. Biochem. J. 336, 19–31. A review that goes into greater depth than this chapter. Metabolic Control Analysis Aiston, S., Hampson, L., Gomez-Foix, A.M., Guinovart, J.J., & Agius, L. (2001) Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis. J. Biol. Chem. 276, 23,858–23,866. Fell, D.A. (1992) Metabolic control analysis: a survey of its theoretical and experimental development. Biochem. J. 286, 313–330. Clear statement of the principles of metabolic control analysis. Fell, D.A. (1997) Understanding the Control of Metabolism, Portland Press, Ltd., London. An excellent, clear exposition of metabolic regulation, from the point of view of metabolic control analysis. If you read only one treatment on metabolic control analysis, this should be it. Jeffrey, F.M.H., Rajagopal, A., Maloy, C.R., & Sherry, A.D. (1991) 13 C-NMR: a simple yet comprehensive method for analysis of intermediary metabolism. Trends Biochem. Sci. 16, 5–10. Brief, intermediate-level review. Kacser, H. & Burns, J.A. (1973) The control of flux. Symp. Soc. Exp. Biol. 32, 65–104. A classic paper in the field. Kacser, H., Burns, J.A., & Fell, D.A. (1995) The control of flux: 21 years on. Biochem. Soc. Trans. 23, 341–366. Schilling, C.H., Schuster, S., Palsson, B.O., & Heinrich, R. (1999) Metabolic pathway analysis: basic concepts and scientific applications in the post-genomic era. Biotechnol. Prog. 15, 296–303. Short, advanced discussion of theoretical treatments that attempt to find ways of manipulating metabolism to optimize the formation of metabolic products. Schuster, S., Fell, D.A., & Dandekar, T. (2000) A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks. Nat. Biotechnol. 18, 326–332. An interesting and provocative analysis of the interplay between the pentose phosphate pathway and glycolysis, from a theoretical standpoint. Shulman, R.G., Block, G., & Rothman, D.L. (1995) In vivo regulation of muscle glycogen synthase and the control of glycogen synthesis. Proc. Natl. Acad. Sci. USA 92, 8535–8542. Review of the use of NMR to measure metabolite concentrations during glycogen synthesis, interpreted by metabolic control analysis. Westerhoff, H.V., Hofmeyr, J.-H.S., & Kholodenko, B.N. (1994) Getting to the inside of cells using metabolic control analysis. Biophys. Chem. 50, 273–283. 1. Measurement of Intracellular Metabolite Con- centrations Measuring the concentrations of metabolic intermediates in a living cell presents great experimental dif- ficulties—usually a cell must be destroyed before metabolite concentrations can be measured. Yet enzymes catalyze meta- bolic interconversions very rapidly, so a common problem associated with these types of measurements is that the findings reflect not the physiological concentrations of metabolites but the equilibrium concentrations. A reliable ex- perimental technique requires all enzyme-catalyzed reactions to be instantaneously stopped in the intact tissue so that the metabolic intermediates do not undergo change. This objec- tive is accomplished by rapidly compressing the tissue be- tween large aluminum plates cooled with liquid nitrogen (H11002190 oC), a process called freeze-clamping. After freezing, which stops enzyme action instantly, the tissue is powdered and the enzymes are inactivated by precipitation with per- chloric acid. The precipitate is removed by centrifugation, and the clear supernatant extract is analyzed for metabolites. To calculate intracellular concentrations, the intracellular vol- ume is determined from the total water content of the tissue and a measurement of the extracellular volume. The intracellular concentrations of the substrates and products of the phosphofructokinase-1 reaction in isolated rat heart tissue are given in the table below. Problems Metabolite Concentration (H9262M) * Fructose 6-phosphate 87.0 Fructose 1,6-bisphosphate 22.0 ATP 11,400 ADP 1,320 Source: From Williamson, J.R. (1965) Glycolytic control mechanisms I: inhibition of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J. Biol. Chem. 240, 2308–2321. * Calculated as μmol/mL of intracellular water. 8885d_c15_560-600 2/26/04 9:04 AM Page 598 mac76 mac76:385_reb: Chapter 15 Problems 599 (a) Calculate Q, [fructose 1,6-bisphosphate] [ADP] / [fructose 6-phosphate][ATP], for the PFK-1 reaction under physiological conditions. (b) Given a H9004GH11032H11034 for the PFK-1 reaction of H1100214.2 kJ/mol, calculate the equilibrium constant for this reaction. (c) Compare the values of Q and KH11032 eq . Is the physiolog- ical reaction near or far from equilibrium? Explain. What does this experiment suggest about the role of PFK-1 as a regula- tory enzyme? 2. Effect of O 2 Supply on Glycolytic Rates The regu- lated steps of glycolysis in intact cells can be identified by studying the catabolism of glucose in whole tissues or organs. For example, the glucose consumption by heart muscle can be measured by artificially circulating blood through an iso- lated intact heart and measuring the concentration of glucose before and after the blood passes through the heart. If the circulating blood is deoxygenated, heart muscle consumes glucose at a steady rate. When oxygen is added to the blood, the rate of glucose consumption drops dramatically, then is maintained at the new, lower rate. Why? 3. Regulation of PFK-1 The effect of ATP on the al- losteric enzyme PFK-1 is shown below. For a given concen- tration of fructose 6-phosphate, the PFK-1 activity increases with increasing concentrations of ATP, but a point is reached beyond which increasing the concentration of ATP inhibits the enzyme. (a) Explain how ATP can be both a substrate and an in- hibitor of PFK-1. How is the enzyme regulated by ATP? (b) In what ways is glycolysis regulated by ATP levels? (c) The inhibition of PFK-1 by ATP is diminished when the ADP concentration is high, as shown in the illustration. How can this observation be explained? 4. Are All Metabolic Reactions at Equilibrium? (a) Phosphoenolpyruvate (PEP) is one of the two phos- phoryl group donors in the synthesis of ATP during glycoly- sis. In human erythrocytes, the steady-state concentration of ATP is 2.24 mM, that of ADP is 0.25 mM, and that of pyruvate is 0.051 mM. Calculate the concentration of PEP at 25 H11034C, as- suming that the pyruvate kinase reaction (see Fig. 13–3) is at equilibrium in the cell. (b) The physiological concentration of PEP in human erythrocytes is 0.023 mM. Compare this with the value ob- tained in (a). Explain the significance of this difference. 5. Cellular Glucose Concentration The concentration of glucose in human blood plasma is maintained at about 5mM. The concentration of free glucose inside a myocyte is much lower. Why is the concentration so low in the cell? What happens to glucose after entry into the cell? Glucose is ad- ministered intravenously as a food source in certain clinical situations. Given that the transformation of glucose to glu- cose 6-phosphate consumes ATP, why not administer intra- venous glucose 6-phosphate instead? 6. Enzyme Activity and Physiological Function The V max of the enzyme glycogen phosphorylase from skeletal muscle is much greater than the V max of the same enzyme from liver tissue. (a) What is the physiological function of glycogen phos- phorylase in skeletal muscle? In liver tissue? (b) Why does the V max of the muscle enzyme need to be greater than that of the liver enzyme? 7. Glycogen Phosphorylase Equilibrium Glycogen phosphorylase catalyzes the removal of glucose from glyco- gen. The H9004GH11032H11034 for this reaction is 3.1 kJ/mol. (a) Calculate the ratio of [P i ] to [glucose 1-phosphate] when the reaction is at equilibrium. (Hint: The removal of glucose units from glycogen does not change the glycogen concentration.) (b) The measured ratio [P i ]/[glucose 1-phosphate] in my- ocytes under physiological conditions is more than 100:1. What does this indicate about the direction of metabolite flow through the glycogen phosphorylase reaction in muscle? (c) Why are the equilibrium and physiological ratios different? What is the possible significance of this difference? 8. Regulation of Glycogen Phosphorylase In muscle tissue, the rate of conversion of glycogen to glucose 6-phos- phate is determined by the ratio of phosphorylase a (active) to phosphorylase b (less active). Determine what happens to the rate of glycogen breakdown if a muscle preparation con- taining glycogen phosphorylase is treated with (a) phospho- rylase kinase and ATP; (b) PP1; (c) epinephrine. 9. Glycogen Breakdown in Rabbit Muscle The intra- cellular use of glucose and glycogen is tightly regulated at four points. In order to compare the regulation of glycolysis when oxygen is plentiful and when it is depleted, consider the utilization of glucose and glycogen by rabbit leg muscle in two physiological settings: a resting rabbit, with low ATP demands, and a rabbit that sights its mortal enemy, the coy- ote, and dashes into its burrow. For each setting, determine the relative levels (high, intermediate, or low) of AMP, ATP, citrate, and acetyl-CoA and how these levels affect the flow of metabolites through glycolysis by regulating specific en- zymes. In periods of stress, rabbit leg muscle produces much of its ATP by anaerobic glycolysis (lactate fermentation) and very little by oxidation of acetyl-CoA derived from fat break- down. 10. Glycogen Breakdown in Migrating Birds Unlike the rabbit with its short dash, migratory birds require energy for extended periods of time. For example, ducks generally fly several thousand miles during their annual migration. The flight muscles of migratory birds have a high oxidative ca- pacity and obtain the necessary ATP through the oxidation of acetyl-CoA (obtained from fats) via the citric acid cycle. Compare the regulation of muscle glycolysis during short- term intense activity, as in the fleeing rabbit, and during ex- tended activity, as in the migrating duck. Why must the reg- ulation in these two settings be different? PFK-1 activity (% of V max ) [ATP] High [ADP] 100 80 60 40 20 0 Low [ADP] 8885d_c15_560-600 2/26/04 9:04 AM Page 599 mac76 mac76:385_reb: Chapter 15 Principles of Metabolic Regulation: Glucose and Glycogen600 11. Enzyme Defects in Carbohydrate Metabo- lism Summaries of four clinical case studies follow. For each case determine which enzyme is defective and des- ignate the appropriate treatment, from the lists provided at the end of the problem. Justify your choices. Answer the questions contained in each case study. (You may need to re- fer to information in Chapter 14.) Case A The patient develops vomiting and diarrhea shortly after milk ingestion. A lactose tolerance test is ad- ministered. (The patient ingests a standard amount of lac- tose, and the glucose and galactose concentrations of blood plasma are measured at intervals. In normal individuals the levels increase to a maximum in about 1 hour, then decline.) The patient’s blood glucose and galactose concentrations do not increase during the test. Why do blood glucose and galac- tose increase and then decrease during the test in normal in- dividuals? Why do they fail to rise in the patient? Case B The patient develops vomiting and diarrhea af- ter ingestion of milk. His blood is found to have a low con- centration of glucose but a much higher than normal con- centration of reducing sugars. The urine test for galactose is positive. Why is the concentration of reducing sugar in the blood high? Why does galactose appear in the urine? Case C The patient complains of painful muscle cramps when performing strenuous physical exercise but has no other symptoms. A muscle biopsy indicates a muscle glyco- gen concentration much higher than normal. Why does glyco- gen accumulate? Case D The patient is lethargic, her liver is enlarged, and a biopsy of the liver shows large amounts of excess glyco- gen. She also has a lower than normal blood glucose level. What is the reason for the low blood glucose in this patient? Defective Enzyme (a) Muscle PFK-1 (b) Phosphomannose isomerase (c) Galactose 1-phosphate uridylyltransferase (d) Liver glycogen phosphorylase (e) Triose kinase (f) Lactase in intestinal mucosa (g) Maltase in intestinal mucosa (h) Muscle-debranching enzyme Treatment 1. Jogging 5 km each day 2. Fat-free diet 3. Low-lactose diet 4. Avoiding strenuous exercise 5. Large doses of niacin (the precursor of NAD H11001 ) 6. Frequent regular feedings 8885d_c15_560-600 2/26/04 9:04 AM Page 600 mac76 mac76:385_reb: chapter A s we saw in Chapter 14, some cells obtain energy (ATP) by fermentation, breaking down glucose in the absence of oxygen. For most eukaryotic cells and many bacteria, which live under aerobic conditions and oxi- dize their organic fuels to carbon dioxide and water, gly- colysis is but the first stage in the complete oxidation of glucose. Rather than being reduced to lactate, ethanol, or some other fermentation product, the pyru- vate produced by glycolysis is further oxidized to H 2 O and CO 2 . This aerobic phase of catabolism is called res- piration. In the broader physiological or macroscopic sense, respiration refers to a multicellular organism’s up- take of O 2 and release of CO 2 . Biochemists and cell bi- ologists, however, use the term in a narrower sense to refer to the molecular processes by which cells consume O 2 and produce CO 2 —processes more precisely termed cellular respiration. Cellular respiration occurs in three major stages (Fig. 16–1). In the first, organic fuel molecules—glu- cose, fatty acids, and some amino acids—are oxidized to yield two-carbon fragments in the form of the acetyl group of acetyl-coenzyme A (acetyl-CoA). In the sec- ond stage, the acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them to CO 2 ; the energy released is conserved in the reduced electron carriers NADH and FADH 2 . In the third stage of respi- ration, these reduced coenzymes are themselves oxidized, giving up protons (H H11001 ) and electrons. The electrons are transferred to O 2 —the final electron acceptor—via a chain of electron-carrying molecules known as the respiratory chain. In the course of elec- tron transfer, the large amount of energy released is conserved in the form of ATP, by a process called ox- idative phosphorylation (Chapter 19). Respiration is more complex than glycolysis and is believed to have evolved much later, after the appearance of cyanobac- teria. The metabolic activities of cyanobacteria account for the rise of oxygen levels in the earth’s atmosphere, a dramatic turning point in evolutionary history. We consider first the conversion of pyruvate to acetyl groups, then the entry of those groups into the citric acid cycle, also called the tricarboxylic acid (TCA) cycle or the Krebs cycle (after its discoverer, Hans Krebs). We next examine the cycle reactions and the enzymes that catalyze them. Because intermediates of the citric acid cycle are also siphoned off as biosyn- thetic precursors, we go on to consider some ways in which these intermediates are replenished. The citric acid cycle is a hub in metabolism, with degradative pathways leading in and anabolic pathways leading out, and it is closely regulated in coordination with other pathways. The chapter ends with a description of the gly- oxylate pathway, a metabolic sequence in some organisms that employs several of the same enzymes and reactions used in the citric acid cycle, bringing about the net syn- thesis of glucose from stored triacylglycerols. THE CITRIC ACID CYCLE 16.1 Production of Acetyl-CoA (Activated Acetate) 602 16.2 Reactions of the Citric Acid Cycle 606 16.3 Regulation of the Citric Acid Cycle 621 16.4 The Glyoxylate Cycle 623 If citrate is added the rate of respiration is often increased . . . the extra oxygen uptake is by far greater than can be accounted for by the complete oxidation of citrate . . . Since citric acid reacts catalytically in the tissue it is probable that it is removed by a primary reaction but regenerated by a subsequent reaction. —H. A. Krebs and W. A. Johnson, article in Enzymologia, 1937 16 601 Hans Krebs, 1900–1981 8885d_c16_601-630 1/27/04 8:54 AM Page 601 mac76 mac76:385_reb: 16.1 Production of Acetyl-CoA (Activated Acetate) In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are ultimately oxidized to CO 2 and H 2 O via the citric acid cycle and the respira- tory chain. Before entering the citric acid cycle, the car- bon skeletons of sugars and fatty acids are degraded to the acetyl group of acetyl-CoA, the form in which the cycle accepts most of its fuel input. Many amino acid carbons also enter the cycle this way, although several amino acids are degraded to other cycle intermediates. Here we focus on how pyruvate, derived from glucose and other sugars by glycolysis, is oxidized to acetyl-CoA and CO 2 by the pyruvate dehydrogenase (PDH) complex, a cluster of enzymes—multiple copies of each of three enzymes—located in the mitochondria of eu- karyotic cells and in the cytosol of prokaryotes. A careful examination of this enzyme complex is re- warding in several respects. The PDH complex is a clas- sic, much-studied example of a multienzyme complex in which a series of chemical intermediates remain bound to the enzyme molecules as a substrate is trans- formed into the final product. Five cofactors, four derived from vitamins, participate in the reaction mech- anism. The regulation of this enzyme complex also illustrates how a combination of covalent modification and allosteric regulation results in precisely regulated flux through a metabolic step. Finally, the PDH complex is the prototype for two other important enzyme com- plexes: H9251-ketoglutarate dehydrogenase, of the citric acid cycle, and the branched-chain H9251-keto acid dehydroge- nase, of the oxidative pathways of several amino acids (see Fig. 18–28). The remarkable similarity in the pro- tein structure, cofactor requirements, and reaction mechanisms of these three complexes doubtless reflects a common evolutionary origin. Pyruvate Is Oxidized to Acetyl-CoA and CO 2 The overall reaction catalyzed by the pyruvate dehy- drogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO 2 Chapter 16 The Citric Acid Cycle602 NADH, FADH 2 (reduced e H11002 carriers) Respiratory (electron-transfer) chain ATPADP + P i H 2 O Stage 3 Electron transfer and oxidative phosphorylation Citric acid cycle Stage 2 Acetyl-CoA oxidation Acetyl-CoA Oxaloacetate CO 2 pyruvate dehydrogenase complex Pyruvate Glycolysis Fatty acids Amino acids e H11002 Stage 1 Acetyl-CoA production 2H + + e H11002 e H11002 e H11002 CO 2 CO 2 e H11002 e H11002 e H11002 e H11002 e H11002 1 2 O 2 Glucose Citrate FIGURE 16–1 Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration. Stage 1: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation of acetyl groups in the citric acid cycle includes four steps in which electrons are abstracted. Stage 3: electrons carried by NADH and FADH 2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane–bound) electron carriers—the respiratory chain— ultimately reducing O 2 to H 2 O. This electron flow drives the produc- tion of ATP. H11001 CoA-SH NADH Acetyl-CoA MD O A C S-CoA NAD H11001 A C Pyruvate CH 3 M P O A O C H11002 O CH 3 CO 2 D H9004GH11032H11034 H11005 H1100233.4 kJ/mol pyruvate dehydrogenase complex (E 1 H11001 E 2 H11001 E 3 ) TPP, lipoate, FAD FIGURE 16–2 Overall reaction catalyzed by the pyruvate dehydro- genase complex. The five coenzymes participating in this reaction, and the three enzymes that make up the enzyme complex, are discussed in the text. 8885d_c16_601-630 1/27/04 8:54 AM Page 602 mac76 mac76:385_reb: and the two remaining carbons become the acetyl group of acetyl-CoA (Fig. 16–2). The NADH formed in this re- action gives up a hydride ion (:H H11002 ) to the respiratory chain (Fig. 16–1), which carries the two electrons to oxygen or, in anaerobic microorganisms, to an alterna- tive electron acceptor such as nitrate or sulfate. The transfer of electrons from NADH to oxygen ultimately generates 2.5 molecules of ATP per pair of electrons. The irreversibility of the PDH complex reaction has been demonstrated by isotopic labeling experiments: the complex cannot reattach radioactively labeled CO 2 to acetyl-CoA to yield carboxyl-labeled pyruvate. The Pyruvate Dehydrogenase Complex Requires Five Coenzymes The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16–2) requires the sequential action of three different en- zymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes de- noted CoA-SH, to emphasize the role of the OSH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nu- trition are vital components of this system: thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pan- tothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers (Chapter 13), and we have encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14–13). Coenzyme A (Fig. 16–3) has a reactive thiol (OSH) group that is critical to the role of CoA as an acyl car- rier in a number of metabolic reactions. Acyl groups are covalently linked to the thiol group, forming thioesters. Because of their relatively high standard free energies of hydrolysis (see Figs 13–6, 13–7), thioesters have a high acyl group transfer potential and can donate their acyl groups to a variety of acceptor molecules. The acyl group attached to coenzyme A may thus be thought of as “activated” for group transfer. The fifth cofactor of the PDH complex, lipoate (Fig. 16–4), has two thiol groups that can undergo reversible oxidation to a disulfide bond (OSOSO), similar to that between two Cys residues in a protein. Because of its capacity to undergo oxidation-reduction reactions, lipoate can serve both as an electron hydro- gen carrier and as an acyl carrier, as we shall see. 16.1 Production of Acetyl-CoA (Activated Acetate) 603 FIGURE 16-3 Coenzyme A (CoA). A hydroxyl group of pantothenic acid is joined to a modified ADP moiety by a phosphate ester bond, and its carboxyl group is attached to H9252-mercaptoethylamine in amide linkage. The hydroxyl group at the 3H11032 position of the ADP moiety has a phosphoryl group not present in free ADP. The OSH group of the mercaptoethylamine moiety forms a thioester with acetate in acetyl- coenzyme A (acetyl-CoA) (lower left). N NH 2 N N N O HH H OH H Reactive thiol group O H11002 O PO 4H11032 1H11032 OH 5H11032 2H11032 O CO O H11002 O O NOCH 2 C PHS CH 3 CCH 2 CH 3 O P H C CH 2 CH 2 O CN H CH 2 -CoA H Ribose 3H11032-phosphate Acetyl-CoA 3H11032-Phosphoadenosine diphosphate Pantothenic acid H9252-Mercapto- ethylamine Coenzyme A Adenine CH 2 O H11002 S O CH 3 O H11002 3H11032 CH 2 CH O CH 2 CH N H CH 3 CH 2 CH 2 CH 2 C S CH 2 CH 2 CH 2 C O S HS HN O CH 2 CH CH 2 CH 2 CH 2 CH 2 CH 2 CH CH 2 CH 2 HS C SHS Lys residue of E 2 Lipoic acid Oxidized form Reduced form Acetylated form Polypeptide chain of E 2 (dihydrolipoyl transacetylase) FIGURE 16–4 Lipoic acid (lipoate) in amide linkage with a Lys residue. The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E 2 of the PDH complex). The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group. 8885d_c16_601-630 1/27/04 8:54 AM Page 603 mac76 mac76:385_reb: The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes The PDH complex contains three enzymes—pyruvate dehydrogenase (E 1 ), dihydrolipoyl transacetylase (E 2 ), and dihydrolipoyl dehydrogenase (E 3 )—each present in multiple copies. The number of copies of each enzyme and therefore the size of the complex varies among species. The PDH complex isolated from mam- mals is about 50 nm in diameter—more than five times the size of an entire ribosome and big enough to be vi- sualized with the electron microscope (Fig. 16–5a). In the bovine enzyme, 60 identical copies of E 2 form a pen- tagonal dodecahedron (the core) with a diameter of about 25 nm (Fig. 16–5b). (The core of the Escherichia coli enzyme contains 24 copies of E 2 .) E 2 is the point of connection for the prosthetic group lipoate, attached through an amide bond to the H9255-amino group of a Lys residue (Fig. 16–4). E 2 has three functionally distinct do- mains (Fig. 16–5c): the amino-terminal lipoyl domain, containing the lipoyl-Lys residue(s); the central E 1 - and E 3 -binding domain; and the inner-core acyltransferase domain, which contains the acyltransferase active site. The yeast PDH complex has a single lipoyl domain with a lipoate attached, but the mammalian complex has two, and E. coli has three (Fig. 16–5c). The domains of E 2 are separated by linkers, sequences of 20 to 30 amino acid residues, rich in Ala and Pro and interspersed with charged residues; these linkers tend to assume their ex- tended forms, holding the three domains apart. The active site of E 1 has bound TPP, and that of E 3 has bound FAD. Also part of the complex are two reg- Chapter 16 The Citric Acid Cycle604 Number of lipoyl domains varies by species. E. coli (3) Mammals (2) Yeast (1) Lipoyl domain Acyltransferase domain (inner core) C N Binding domain (involved in E 2 -E 1 and E 2 -E 3 binding) Flexible polypeptide linker 50 nm FIGURE 16–5 Structure of the pyruvate dehydrogenase complex (a) Cryoelectron micrograph of PDH complexes isolated from bovine kidney. In cryoelectron microscopy, biological samples are viewed at extremely low temperatures; this avoids potential artifacts introduced by the usual process of dehydrating, fixing, and staining. (b) Three- dimensional image of PDH complex, showing the subunit structure: E 1 , pyruvate dehydrogenase; E 2 , dihydrolipoyl transacetylase; and E 3 , dihydrolipoyl dehydrogenase. This image is reconstructed by analysis of a large number of images such as those in (a), combined with crys- tallographic studies of individual subunits. The core (green) consists of 60 molecules of E 2 , arranged in 20 trimers to form a pentagonal dodecahedron. The lipoyl domain of E 2 (blue) reaches outward to touch the active sites of E 1 molecules (yellow) arranged on the E 2 core. A number of E 3 subunits (red) are also bound to the core, where the swinging arm on E 2 can reach their active sites. An asterisk marks the site where a lipoyl group is attached to the lipoyl domain of E 2 . To make the structure clearer, about half of the complex has been cut away from the front. This model was prepared by Z. H. Zhou et al. (2001); in another model, proposed by J. L. S. Milne et al. (2002), the E 3 subunits are located more toward the periphery (see Further Read- ing). (c) E 2 consists of three types of domains linked by short polypep- tide linkers: a catalytic acyltransferase domain; a binding domain, in- volved in the binding of E 2 to E 1 and E 3 ; and one or more (depending on the species) lipoyl domains. E 1 E 2 E 3 10 nm * (a) (b) (c) 8885d_c16_604 1/30/04 11:46 AM Page 604 mac76 mac76:385_reb: ulatory proteins, a protein kinase and a phosphoprotein phosphatase, discussed below. This basic E 1 -E 2 -E 3 structure has been conserved during evolution and used in a number of similar metabolic reactions, including the oxidation of H9251-ketoglutarate in the citric acid cycle (de- scribed below) and the oxidation of H9251-keto acids derived from the breakdown of the branched-chain amino acids valine, isoleucine, and leucine (see Fig. 18–28). Within a given species, E 3 of PDH is identical to E 3 of the other two enzyme complexes. The attachment of lipoate to the end of a Lys side chain in E 2 produces a long, flex- ible arm that can move from the active site of E 1 to the active sites of E 2 and E 3 , a distance of perhaps 5 nm or more. In Substrate Channeling, Intermediates Never Leave the Enzyme Surface Figure 16–6 shows schematically how the pyruvate de- hydrogenase complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate. Step 1 is essentially identical to the reac- tion catalyzed by pyruvate decarboxylase (see Fig. 14–13c); C-1 of pyruvate is released as CO 2 , and C-2, which in pyruvate has the oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group. This first step is the slowest and therefore limits the rate of the overall reaction. It is also the point at which the PDH complex exercises its substrate specificity. In step 2 the hydroxyethyl group is oxidized to the level of a car- boxylic acid (acetate). The two electrons removed in this reaction reduce the OSOSO of a lipoyl group on E 2 to two thiol (OSH) groups. The acetyl moiety produced in this oxidation-reduction reaction is first esterified to one of the lipoyl OSH groups, then trans- esterified to CoA to form acetyl-CoA (step 3 ). Thus the energy of oxidation drives the formation of a high- energy thioester of acetate. The remaining reactions catalyzed by the PDH complex (by E 3 , in steps 4 and 5 ) are electron transfers necessary to regenerate the oxidized (disulfide) form of the lipoyl group of E 2 to prepare the enzyme complex for another round of oxidation. The electrons removed from the hydrox- yethyl group derived from pyruvate pass through FAD to NAD H11001 . Central to the mechanism of the PDH complex are the swinging lipoyllysyl arms of E 2 , which accept from E 1 the two electrons and the acetyl group derived from pyruvate, passing them to E 3 . All these enzymes and coenzymes are clustered, allowing the intermediates to react quickly without diffusing away from the sur- face of the enzyme complex. The five-reaction se- quence shown in Figure 16–6 is thus an example of substrate channeling. The intermediates of the multistep sequence never leave the complex, and the local concentration of the substrate of E 2 is kept very high. Channeling also prevents theft of the activated acetyl group by other enzymes that use this group as substrate. As we shall see, a similar tethering mecha- nism for the channeling of substrate between active 16.1 Production of Acetyl-CoA (Activated Acetate) 605 Acyl lipoyllysine Oxidized lipoyllysine Reduced lipoyllysine CoA-SH TPP CO 2 Pyruvate Hydroxyethyl TPP Pyruvate dehydrogenase, E 1 Dihydrolipoyl transacetylase, E 2 Dihydrolipoyl dehydrogenase, E 3 TPP Lys Acetyl-CoA O CH 3 CoAS-C O CH 3 C O O – C CHOH CH 3 FAD FADH 2 SH SH NADH + H + NAD + 3 4 5 21 S S C CH 3 S SH O FIGURE 16–6 Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex. The fate of pyruvate is traced in red. In step H220711 pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydrogenase (E 1 ), undergoing decarboxylation to the hy- droxyethyl derivative (see Fig. 14–13). Pyruvate dehydrogenase also carries out step H220712 , the transfer of two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core en- zyme, dihydrolipoyl transacetylase (E 2 ), to form the acetyl thioester of the reduced lipoyl group. Step H220713 is a transesterification in which the OSH group of CoA replaces the OSH group of E 2 to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group. In step H220714di- hydrolipoyl dehydrogenase (E 3 ) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E 2 to the FAD prosthetic group of E 3 , restoring the oxidized form of the lipoyllysyl group of E 2 . In step H220715 the reduced FADH 2 of E 3 transfers a hydride ion to NAD H11001 , forming NADH. The enzyme complex is now ready for another cat- alytic cycle. (Subunit colors correspond to those in Fig. 16–5b.) 8885d_c16_601-630 1/27/04 8:54 AM Page 605 mac76 mac76:385_reb: sites is used in some other enzymes, with lipoate, bi- otin, or a CoA-like moiety serving as cofactors. As one might predict, mutations in the genes for the subunits of the PDH complex, or a dietary thiamine deficiency, can have severe consequences. Thiamine-deficient animals are unable to oxidize pyru- vate normally. This is of particular importance to the brain, which usually obtains all its energy from the aer- obic oxidation of glucose in a pathway that necessarily includes the oxidation of pyruvate. Beriberi, a disease that results from thiamine deficiency, is characterized by loss of neural function. This disease occurs primarily in populations that rely on a diet consisting mainly of white (polished) rice, which lacks the hulls in which most of the thiamine of rice is found. People who ha- bitually consume large amounts of alcohol can also de- velop thiamine deficiency, because much of their dietary intake consists of the vitamin-free “empty calories” of distilled spirits. An elevated level of pyruvate in the blood is often an indicator of defects in pyruvate oxi- dation due to one of these causes. ■ SUMMARY 16.1 Production of Acetyl-CoA (Activated Acetate) ■ Pyruvate, the product of glycolysis, is converted to acetyl-CoA, the starting material for the citric acid cycle, by the pyruvate dehydrogenase complex. ■ The PDH complex is composed of multiple copies of three enzymes: pyruvate dehydrogenase, E 1 (with its bound cofactor TPP); dihydrolipoyl transacetylase, E 2 (with its covalently bound lipoyl group); and dihydrolipoyl dehydrogenase, E 3 (with its cofactors FAD and NAD). ■ E 1 catalyzes first the decarboxylation of pyruvate, producing hydroxyethyl-TPP, and then the oxidation of the hydroxyethyl group to an acetyl group. The electrons from this oxidation reduce the disulfide of lipoate bound to E 2 , and the acetyl group is transferred into thioester linkage with one OSH group of reduced lipoate. ■ E 2 catalyzes the transfer of the acetyl group to coenzyme A, forming acetyl-CoA. ■ E 3 catalyzes the regeneration of the disulfide (oxidized) form of lipoate; electrons pass first to FAD, then to NAD H11001 . ■ The long lipoyllysine arm swings from the active site of E 1 to E 2 to E 3 , tethering the intermediates to the enzyme complex to allow substrate channeling. ■ The organization of the PDH complex is very similar to that of the enzyme complexes that catalyze the oxidation of H9251-ketoglutarate and the branched-chain H9251-keto acids. 16.2 Reactions of the Citric Acid Cycle We are now ready to trace the process by which acetyl- CoA undergoes oxidation. This chemical transformation is carried out by the citric acid cycle, the first cyclic pathway we have encountered (Fig. 16–7). To begin a turn of the cycle, acetyl-CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate. Citrate is then transformed into isocitrate, also a six-carbon molecule, which is dehy- drogenated with loss of CO 2 to yield the five-carbon compound H9251-ketoglutarate (also called oxoglutarate). H9251-Ketoglutarate undergoes loss of a second molecule of CO 2 and ultimately yields the four-carbon compound succinate. Succinate is then enzymatically converted in three steps into the four-carbon oxaloacetate—which is then ready to react with another molecule of acetyl-CoA. In each turn of the cycle, one acetyl group (two carbons) enters as acetyl-CoA and two molecules of CO 2 leave; one molecule of oxaloacetate is used to form citrate and one molecule of oxaloacetate is regenerated. No net removal of oxaloacetate occurs; one molecule of oxalo- acetate can theoretically bring about oxidation of an in- finite number of acetyl groups, and, in fact, oxaloacetate is present in cells in very low concentrations. Four of the eight steps in this process are oxidations, in which the energy of oxidation is very efficiently conserved in the form of the reduced coenzymes NADH and FADH 2 . As noted earlier, although the citric acid cycle is central to energy-yielding metabolism its role is not lim- ited to energy conservation. Four- and five-carbon in- termediates of the cycle serve as precursors for a wide variety of products. To replace intermediates removed for this purpose, cells employ anaplerotic (replenishing) reactions, which are described below. Eugene Kennedy and Albert Lehninger showed in 1948 that, in eukaryotes, the entire set of reactions of the citric acid cycle takes place in mitochondria. Iso- lated mitochondria were found to contain not only all the enzymes and coenzymes required for the citric acid cycle, but also all the enzymes and proteins necessary for the last stage of respiration—electron transfer and ATP synthesis by oxidative phosphorylation. As we shall see in later chapters, mitochondria also contain the en- zymes for the oxidation of fatty acids and some amino acids to acetyl-CoA, and the oxidative degradation of other amino acids to H9251-ketoglutarate, succinyl-CoA, or oxaloacetate. Thus, in nonphotosynthetic eukaryotes, the mitochondrion is the site of most energy-yielding Chapter 16 The Citric Acid Cycle606 8885d_c16_601-630 1/27/04 8:54 AM Page 606 mac76 mac76:385_reb: 16.2 Reactions of the Citric Acid Cycle 607 CH 3 C O S-CoA H 2 O CoA-SH CH 2 COO H11002 HO H C O CoA-SH H 2 O COO H11002 C COO H11002 CH CH 2 COO H11002 Oxaloacetate Acetyl-CoA C O CH 2 COO H11002 COO H11002 C COO H11002 CH 2 COO H11002 Malate C H HO CCOO H11002 CH 2 COO H11002 Citrate C COO H11002 H CH 2 COO H11002 CH 2 COO H11002 Isocitrate COO H11002 O CO 2 COO H11002 C CH 2 CH 2 COO H11002 Succinyl-CoA CH 2 COO H11002 COO H11002 HC HO COO H11002 Succinate COO H11002 CH Fumarate aconitase fumarase aconitase CH 2 2b 2a 1 3 45 6 7 8 Condensation Dehydration Hydration Dehydrogenation Hydration Dehydrogenation CO 2 S-CoA CoA-SH CH 2 H 2 O H 2 O NADH Citric acid cycle malate dehydrogenase citrate synthase isocitrate dehydrogenase H9251-ketoglutarate dehydrogenase complex succinyl-CoA synthetase succinate dehydrogenase GTP (ATP) Substrate-level phosphorylation Oxidative decarboxylation GDP (ADP) H11001 P i H9251-Ketoglutarate Oxidative decarboxylation cis-Aconitate FADH 2 FIGURE 16–7 Reactions of the citric acid cycle. The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are not the carbons released as CO 2 in the first turn. Note that in succinate and fumarate, the two-carbon group derived from acetate can no longer be specifically denoted; because succinate and fumarate are symmetric molecules, C-1 and C-2 are indistinguishable from C-4 and C-3. The number beside each reaction step corresponds to a numbered heading on pages 608–612. The red arrows show where energy is conserved by electron transfer to FAD or NAD H11001 , forming FADH 2 or NADH H11001 H H11001 . Steps H220711,H220713, and H220714 are essentially irreversible in the cell; all other steps are re- versible. The product of step H220715 may be either ATP or GTP, depend- ing on which succinyl-CoA synthetase isozyme is the catalyst. 8885d_c16_601-630 1/27/04 8:54 AM Page 607 mac76 mac76:385_reb: oxidative reactions and of the coupled synthesis of ATP. In photosynthetic eukaryotes, mitochondria are the ma- jor site of ATP production in the dark, but in daylight chloroplasts produce most of the organism’s ATP. In most prokaryotes, the enzymes of the citric acid cycle are in the cytosol, and the plasma membrane plays a role analogous to that of the inner mitochondrial mem- brane in ATP synthesis (Chapter 19). The Citric Acid Cycle Has Eight Steps In examining the eight successive reaction steps of the citric acid cycle, we place special emphasis on the chem- ical transformations taking place as citrate formed from acetyl-CoA and oxaloacetate is oxidized to yield CO 2 and the energy of this oxidation is conserved in the reduced coenzymes NADH and FADH 2 . 1 Formation of Citrate The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase: In this reaction the methyl carbon of the acetyl group is joined to the carbonyl group (C-2) of oxaloacetate. Citroyl-CoA is a transient intermediate formed on the active site of the enzyme (see Fig. 16–9). It rapidly undergoes hydrolysis to free CoA and citrate, which are released from the active site. The hydrolysis of this high-energy thioester intermediate makes the forward reaction highly exergonic. The large, negative standard free-energy change of the citrate synthase reaction is essential to the operation of the cycle because, as noted earlier, the concentration of oxaloacetate is normally very low. The CoA liberated in this reaction is recycled to participate in the oxidative decarboxylation of an- other molecule of pyruvate by the PDH complex. Citrate synthase from mitochondria has been crys- tallized and visualized by x-ray diffraction in the pres- ence and absence of its substrates and inhibitors (Fig. 16–8). Each subunit of the homodimeric enzyme is a single polypeptide with two domains, one large and rigid, the other smaller and more flexible, with the ac- tive site between them. Oxaloacetate, the first substrate to bind to the enzyme, induces a large conformational citrate synthaseS-CoA H11001 CoA-SH COO H11002 O C COO H11002 H 2 O CH 2 Acetyl-CoA CH 3 C O Oxaloacetate Citrate HO COO H11002 C COO H11002 CH 2 O H11002 O CCH 2 H9004GH11032H11034 H11005 H1100232.2 kJ/mol change in the flexible domain, creating a binding site for the second substrate, acetyl-CoA. When citroyl-CoA has formed in the enzyme active site, another conforma- tional change brings about thioester hydrolysis, releas- ing CoA-SH. This induced fit of the enzyme first to its substrate and then to its reaction intermediate de- creases the likelihood of premature and unproductive cleavage of the thioester bond of acetyl-CoA. Kinetic studies of the enzyme are consistent with this ordered bisubstrate mechanism (see Fig. 6–13). The reaction catalyzed by citrate synthase is essentially a Claisen con- densation (p. 485), involving a thioester (acetyl-CoA) and a ketone (oxaloacetate) (Fig. 16–9). 2 Formation of Isocitrate via cis-Aconitate The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate, which normally does Chapter 16 The Citric Acid Cycle608 (b) (a) FIGURE 16–8 Structure of citrate synthase. The flexible domain of each subunit undergoes a large conformational change on binding oxaloacetate creating a binding site for acetyl-CoA. (a) open form of the enzyme alone (PDB ID 5CSC); (b) closed form with bound oxaloacetate (yellow) and a stable analog of acetyl-CoA (carboxymethyl- CoA; red) (derived from PDB ID 5CTS). 8885d_c16_608 1/30/04 11:46 AM Page 608 mac76 mac76:385_reb: not dissociate from the active site. Aconitase can pro- mote the reversible addition of H 2 O to the double bond of enzyme-bound cis-aconitate in two different ways, one leading to citrate and the other to isocitrate: Although the equilibrium mixture at pH 7.4 and 25 H11034C contains less than 10% isocitrate, in the cell the reac- tion is pulled to the right because isocitrate is rapidly consumed in the next step of the cycle, lowering its steady-state concentration. Aconitase contains an iron- sulfur center (Fig. 16–10), which acts both in the bind- ing of the substrate at the active site and in the catalytic addition or removal of H 2 O. 16.2 Reactions of the Citric Acid Cycle 609 O – + O H N H N Asp 375Citrate synthase His 274 O H O Asp 375 O O H 2 C C COO – HC H H CoA Acetyl-CoA The thioester linkage in acetyl-CoA activates the methyl hydrogens, and Asp 375 abstracts a proton from the methyl group, forming an enolate intermediate. The intermediate is stabilized by hydrogen bonding to and/or protonation by His 274 (full protonation is shown). The enol(ate) rearranges to attack the carbonyl carbon of oxaloacetate, with His 274 positioned to abstract the proton it had previously donated. His 320 acts as a general acid. The thioester is subsequently hydrolyzed, regenerating CoA-SH and producing citrate. The resulting condensation generates citroyl-CoA. Citroyl-CoA S-C O HC H C CoA CoA-SH S-C CH 2 : : + H N H N His 320 H O HC H CoA Enol intermediate S-C COO – Oxaloacetate H 2 C CO COO – COO – CH 2 H 2 O COO – Citrate COO – HC H C COO – HO COO – COO – H N H N His 274 HO + N H N His 320 N H H N His 274 N H N His 320 1 3 2 His 274 His 320 Asp 375 Asp 375 MECHANISM FIGURE 16–9 Citrate synthase. In the mammalian cit- rate synthase reaction, oxaloacetate binds first, in a strictly ordered re- action sequence. This binding triggers a conformation change that opens up the binding site for acetyl-CoA. Oxaloacetetate is specifically oriented in the active site of citrate synthase by interaction of its two carboxylates with two positively charged Arg residues (not shown here). The details of the mechanism are described in the figure. Citrate Synthase Mechanism HO H 2 O H CH 2 COO H11002 HC COO H11002 C COO H11002 H 2 O Isocitrate aconitase CH 2 COO H11002 H C COO H11002 C COO H11002 HO H CH 2 COO H11002 H C COO H11002 C COO H11002 Citrate aconitase H9004GH11032H11034 H11005 13.3 kJ/mol cis-Aconitate 8885d_c16_609 1/30/04 11:46 AM Page 609 mac76 mac76:385_reb: 3 Oxidation of Isocitrate to H9251-Ketoglutarate and CO 2 In the next step, isocitrate dehydrogenase catalyzes oxida- tive decarboxylation of isocitrate to form H9251-ketoglu- tarate (Fig. 16–11). Mn 2H11001 in the active site interacts with the carbonyl group of the intermediate oxalosucci- nate, which is formed transiently but does not leave the binding site until decarboxylation converts it to H9251- ketoglutarate. Mn 2H11001 also stabilizes the enol formed tran- siently by decarboxylation. There are two different forms of isocitrate dehy- drogenase in all cells, one requiring NAD H11001 as electron acceptor and the other requiring NADP H11001 . The overall reactions are otherwise identical. In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle. The main func- tion of the NADP-dependent enzyme, found in both the mitochondrial matrix and the cytosol, may be the gen- eration of NADPH, which is essential for reductive an- abolic reactions. 4 Oxidation of H9251-Ketoglutarate to Succinyl-CoA and CO 2 The next step is another oxidative decarboxylation, in which H9251-ketoglutarate is converted to succinyl-CoA and CO 2 by the action of the H9251-ketoglutarate dehy- drogenase complex; NAD H11001 serves as electron accep- tor and CoA as the carrier of the succinyl group. The energy of oxidation of H9251-ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA: This reaction is virtually identical to the pyruvate dehydrogenase reaction discussed above, and the H9251-ketoglutarate dehydrogenase complex closely resem- bles the PDH complex in both structure and function. It includes three enzymes, homologous to E 1 , E 2 , and E 3 of the PDH complex, as well as enzyme-bound TPP, bound lipoate, FAD, NAD, and coenzyme A. Both com- plexes are certainly derived from a common evolution- ary ancestor. Although the E 1 components of the two complexes are structurally similar, their amino acid se- quences differ and, of course, they have different bind- ing specificities: E 1 of the PDH complex binds pyruvate, and E 1 of the H9251-ketoglutarate dehydrogenase complex binds H9251-ketoglutarate. The E 2 components of the two complexes are also very similar, both having covalently bound lipoyl moieties. The subunits of E 3 are identical in the two enzyme complexes. Chapter 16 The Citric Acid Cycle610 COO H11002 CH 2 H H C C HO Isocitrate Oxalosuccinate a-Ketoglutarate NAD(P) H11001 NAD(P)H H11001 H H11001 H H11001 isocitrate dehydrogenase Mn 2H11001 C O H11002 C O O H11002 O COO H11002 CH 2 CO 2 H O C C C O H11002 C O O H11002 O COO H11002 CH 2 H O C H C C O H11002 O Mn 2H11001 COO H11002 CH 2 H C C O H11002 C O H11002 O 1 2 3 MECHANISM FIGURE 16–11 Isocitrate dehydrogenase. In this reac- tion, the substrate, isocitrate, loses one carbon by oxidative decar- boxylation. In step H220711 , isocitrate binds to the enzyme and is oxidized by hydride transfer to NAD + or NADP + , depending on the isocitrate dehydrogenase isozyme. (See Fig. 14–12 for more information on hy- dride transfer reactions involving NAD + and NADP + .) The resulting carbonyl group sets up the molecule for decarboxylation in step H220712 . Interaction of the carbonyl oxygen with a bound Mn 2+ ion increases the electron-withdrawing capacity of the carbonyl group and fac- ilitates the decarboxylation step. The reaction is completed in step H220713 by rearrangement of the enol intermediate to generate H9251-ketoglu- tarate. S Fe O H O H C CH 2 COO H11002 H11002 OOC C C H O O H H S Fe Fe S Fe S Citrate S SCys SCys Cys B FIGURE 16–10 Iron-sulfur center in aconitase. The iron-sulfur center is in red, the citrate molecule in blue. Three Cys residues of the enzyme bind three iron atoms; the fourth iron is bound to one of the carboxyl groups of citrate and also interacts noncovalently with a hydroxyl group of citrate (dashed bond). A basic residue (:B) on the enzyme helps to position the citrate in the active site. The iron-sulfur center acts in both substrate binding and catalysis. The general properties of iron-sulfur proteins are discussed in Chapter 19 (see Fig. 19–5). C O S-CoA CH 2 CH 2 COO H11002 H9251-ketoglutarate dehydrogenase complex CoA-SH NAD H11001 NADH COO H11002 CO CH 2 CH 2 COO H11002 H9251-Ketoglutarate Succinyl-CoA CO 2H11001 H9004GH11032H11034 H11005 H1100233.5 kJ/mol 8885d_c16_610 1/30/04 11:47 AM Page 610 mac76 mac76:385_reb: 5 Conversion of Succinyl-CoA to Succinate Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis (H9004GH11032H11034 ≈ H1100236 kJ/mol). In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in ei- ther GTP or ATP, with a net H9004GH11032H11034 of only H110022.9 kJ/mol. Succinate is formed in the process: The enzyme that catalyzes this reversible reaction is called succinyl-CoA synthetase or succinic thioki- nase; both names indicate the participation of a nucle- oside triphosphate in the reaction (Box 16–1). This energy-conserving reaction involves an inter- mediate step in which the enzyme molecule itself be- comes phosphorylated at a His residue in the active site (Fig. 16–12a). This phosphoryl group, which has a high group transfer potential, is transferred to ADP (or GDP) to form ATP (or GTP). Animal cells have two isozymes of succinyl-CoA synthetase, one specific for ADP and the other for GDP. The enzyme has two subunits, H9251 (M r 32,000), which has the P -His residue (His 246 ) and the binding site for CoA, and H9252 (M r 42,000), which confers specificity for either ADP or GDP. The active site is at the interface between subunits. The crystal structure of succinyl-CoA synthetase reveals two “power helices” (one from each subunit), oriented so that their electric dipoles situate partial positive charges close to the negatively charged P -His (Fig. 16–12b), stabilizing the phosphoenzyme intermediate. (Recall the similar role of helix dipoles in stabilizing K H11001 ions in the K H11001 channel (see Fig. 11–48).) 16.2 Reactions of the Citric Acid Cycle 611 FIGURE 16–12 The succinyl-CoA synthetase reaction. (a) In step H220711 a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. In step H220712 the suc- cinyl phosphate donates its phosphoryl group to a His residue on the enzyme, forming a high-energy phosphohistidyl enzyme. In step H220713 the phosphoryl group is transferred from the His residue to the termi- nal phosphate of GDP (or ADP), forming GTP (or ATP). (b) Succinyl- CoA synthetase of E. coli (derived from PDB ID 1SCU). The bacterial and mammalian enzymes have similar amino acid sequences and pre- sumably have very similar three-dimensional structures. The active site includes part of both the H9251 (blue) and H9252 (brown) subunits. The power helices (bright blue, dark brown) situate the partial positive charges of the helix dipole near the phosphate group (orange) on His 246 of the H9251 chain, stabilizing the phosphohistidyl enzyme. Coenzyme A is shown here as a red stick structure. (To improve the visibility of the power he- lices, some nearby secondary structures have been made transparent.) His His His His CH 2 CH 2 O C S-CoA CH 2 C O O H11002 Succinyl-CoA Succinyl-CoA synthetase O O H11002 C CH 2 Enzyme-bound succinyl phosphate O C O P 2 1 Succinate CH 2 O CCH 2 C O H11002 O O H11002 Phosphohistidyl enzyme 3 CoA-SH GDP GTP P i P (a) (b) H9004GH11032H11034 H11005 H110022.9 kJ/mol S-CoA CH 2 COO H11002 C O CH 2 CH 2 COO H11002 Succinyl-CoA CH 2 COO H11002 Succinate succinyl-CoA synthetase CoA-SHGTPGDP H11001 P i 8885d_c16_601-630 1/27/04 8:54 AM Page 611 mac76 mac76:385_reb: The formation of ATP (or GTP) at the expense of the energy released by the oxidative decarboxylation of H9251-ketoglutarate is a substrate-level phosphorylation, like the synthesis of ATP in the glycolytic reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and pyru- vate kinase (see Fig. 14–2). The GTP formed by succinyl- CoA synthetase can donate its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase (p. 505): GTP H11001 ADP On GDP H11001 ATP H9004GH11032H11034 H11005 0 kJ/mol Thus the net result of the activity of either isozyme of succinyl-CoA synthetase is the conservation of energy as ATP. There is no change in free energy for the nu- cleoside diphosphate kinase reaction; ATP and GTP are energetically equivalent. 6 Oxidation of Succinate to Fumarate The succinate formed from succinyl-CoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase: In eukaryotes, succinate dehydrogenase is tightly bound to the inner mitochondrial membrane; in prokaryotes, to the plasma membrane. The enzyme contains three dif- ferent iron-sulfur clusters and one molecule of covalently bound FAD (see Fig. 19–xx). Electrons pass from suc- cinate through the FAD and iron-sulfur centers before entering the chain of electron carriers in the mitochon- drial inner membrane (or the plasma membrane in bac- teria). Electron flow from succinate through these car- riers to the final electron acceptor, O 2 , is coupled to the synthesis of about 1.5 ATP molecules per pair of elec- trons (respiration-linked phosphorylation). Malonate, an analog of succinate not normally present in cells, is a strong competitive inhibitor of succinate dehydroge- nase and its addition to mitochondria blocks the activ- ity of the citric acid cycle. 7 Hydration of Fumarate to Malate The reversible hydra- tion of fumarate to L-malate is catalyzed by fumarase C O CH 2 Succinate C OO H11002 O H11002 C O CH 2 Malonate C OO H11002 O H11002 CH 2 (formally, fumarate hydratase). The transition state in this reaction is a carbanion: Chapter 16 The Citric Acid Cycle612 H9004GH11032H11034 H11005 0 kJ/mol COO H11002 Succinate succinate dehydrogenase FAD H 2 CH 2 Fumarate CH 2 COO H11002 FAD OOC H11002 COO H11002 C C H H H9004GH11032H11034 H11005 29.7 kJ/mol C CH 2 COO H11002 malate dehydrogenase NAD H11001 NADH H11001 H H11001 O L-Malate Oxaloacetate COO H11002 COO H11002 C CH 2 COO H11002 HHO H9004GH11032H11034 H11005 H110023.8 kJ/mol Carbanion transition state fumarase OH H11002 fumarase H H11001 Fumarate OOC H11002 COO H11002 C C H H OH OOC H11002 C C COO H11002 H11002 H H OH OOC H11002 C C COO H11002 H H H Malate 8 Oxidation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, NAD-linked L-malate dehy- drogenase catalyzes the oxidation of L-malate to ox- aloacetate: Fumarate C COO H11002 Maleate CH 2 H11002 OOC D-MalateL-Malate H H H C C COO H11002 H COO H11002 C COH H COO H11002 COO H11002 CH 2 HO COO H11002 CH COO H11002 The equilibrium of this reaction lies far to the left under standard thermodynamic conditions, but in intact cells This enzyme is highly stereospecific; it catalyzes hydra- tion of the trans double bond of fumarate but not the cis double bond of maleate (the cis isomer of fumarate). In the reverse direction (from L-malate to fumarate), fuma- rase is equally stereospecific: D-malate is not a substrate. 8885d_c16_612 1/30/04 11:47 AM Page 612 mac76 mac76:385_reb: oxaloacetate is continually removed by the highly exer- gonic citrate synthase reaction (step 1 of Fig. 16–7). This keeps the concentration of oxaloacetate in the cell extremely low (H1102110 H110026 M), pulling the malate dehydro- genase reaction toward the formation of oxaloacetate. Although the individual reactions of the citric acid cycle were initially worked out in vitro, using minced muscle tissue, the pathway and its regulation have also been studied extensively in vivo. By using radioactively la- beled precursors such as [ 14 C]pyruvate and [ 14 C]acetate, researchers have traced the fate of individual carbon atoms through the citric acid cycle. Some of the earliest experiments with isotopes produced an unexpected re- sult, however, which aroused considerable controversy about the pathway and mechanism of the citric acid cy- cle. In fact, these experiments at first seemed to show that citrate was not the first tricarboxylic acid to be formed. Box 16–2 gives some details of this episode in the history of citric acid cycle research. Metabolic flux 16.2 Reactions of the Citric Acid Cycle 613 BOX 16–1 WORKING IN BIOCHEMISTRY Synthases and Synthetases; Ligases and Lyases; Kinases, Phosphatases, and Phosphorylases: Yes, the Names Are Confusing! Citrate synthase is one of many enzymes that catalyze condensation reactions, yielding a product more chem- ically complex than its precursors. Synthases catalyze condensation reactions in which no nucleoside triphos- phate (ATP, GTP, and so forth) is required as an en- ergy source. Synthetases catalyze condensations that do use ATP or another nucleoside triphosphate as a source of energy for the synthetic reaction. Succinyl- CoA synthetase is such an enzyme. Ligases (from the Latin ligare, “to tie together”) are enzymes that cat- alyze condensation reactions in which two atoms are joined using ATP or another energy source. (Thus syn- thetases are ligases.) DNA ligase, for example, closes breaks in DNA molecules, using energy supplied by ei- ther ATP or NAD H11001 ; it is widely used in joining DNA pieces for genetic engineering. Ligases are not to be confused with lyases, enzymes that catalyze cleavages (or, in the reverse direction, additions) in which elec- tronic rearrangements occur. The PDH complex, which oxidatively cleaves CO 2 from pyruvate, is a member of the large class of lyases. The name kinase is applied to enzymes that transfer a phosphoryl group from a nucleoside triphos- phate such as ATP to an acceptor molecule—a sugar (as in hexokinase and glucokinase), a protein (as in glycogen phosphorylase kinase), another nucleotide (as in nucleoside diphosphate kinase), or a metabolic intermediate such as oxaloacetate (as in PEP car- boxykinase). The reaction catalyzed by a kinase is a phosphorylation. On the other hand, phosphoroly- sis is a displacement reaction in which phosphate is the attacking species and becomes covalently at- tached at the point of bond breakage. Such reactions are catalyzed by phosphorylases. Glycogen phos- phorylase, for example, catalyzes the phosphorolysis of glycogen, producing glucose 1-phosphate. Dephos- phorylation, the removal of a phosphoryl group from a phosphate ester, is catalyzed by phosphatases, with water as the attacking species. Fructose bis- phosphatase-1 converts fructose 1,6-bisphosphate to fructose 6-phosphate in gluconeogenesis, and phos- phorylase a phosphatase removes phosphoryl groups from phosphoserine in phosphorylated glycogen phosphorylase. Whew! Unfortunately, these descriptions of enzyme types overlap, and many enzymes are commonly called by two or more names. Succinyl-CoA synthetase, for ex- ample, is also called succinate thiokinase; the enzyme is both a synthetase in the citric acid cycle and a ki- nase when acting in the direction of succinyl-CoA syn- thesis. This raises another source of confusion in the naming of enzymes. An enzyme may have been dis- covered by the use of an assay in which, say, A is con- verted to B. The enzyme is then named for that reac- tion. Later work may show, however, that in the cell, the enzyme functions primarily in converting B to A. Commonly, the first name continues to be used, al- though the metabolic role of the enzyme would be bet- ter described by naming it for the reverse reaction. The glycolytic enzyme pyruvate kinase illustrates this situation (p. 532). To a beginner in biochemistry, this duplication in nomenclature can be bewildering. In- ternational committees have made heroic efforts to systematize the nomenclature of enzymes (see Table 6–3 for a brief summary of the system), but some sys- tematic names have proved too long and cumbersome and are not frequently used in biochemical conversa- tion. We have tried throughout this book to use the en- zyme name most commonly used by working bio- chemists and to point out cases in which an enzyme has more than one widely used name. For current in- formation on enzyme nomenclature, refer to the rec- ommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Bi- ology (www.chem.qmw.ac.uk/iubmb/nomenclature/). 8885d_c16_601-630 1/27/04 8:54 AM Page 613 mac76 mac76:385_reb: through the cycle can now be monitored in living tis- sue by using 13 C-labeled precursors and whole-tissue NMR spectroscopy. Because the NMR signal is unique to the compound containing the 13 C, biochemists can trace the movement of precursor carbons into each cycle intermediate and into compounds derived from the intermediates. This technique has great promise for studies of regulation of the citric acid cycle and its interconnections with other metabolic pathways such as glycolysis. The Energy of Oxidations in the Cycle Is Efficiently Conserved We have now covered one complete turn of the citric acid cycle (Fig. 16–13). A two-carbon acetyl group en- tered the cycle by combining with oxaloacetate. Two carbon atoms emerged from the cycle as CO 2 from the oxidation of isocitrate and H9251-ketoglutarate. The energy released by these oxidations was conserved in the re- duction of three NAD H11001 and one FAD and the produc- tion of one ATP or GTP. At the end of the cycle a mol- ecule of oxaloacetate was regenerated. Note that the two carbon atoms appearing as CO 2 are not the same two carbons that entered in the form of the acetyl group; additional turns around the cycle are required to release these carbons as CO 2 (Fig. 16–7). Although the citric acid cycle directly generates only one ATP per turn (in the conversion of succinyl- CoA to succinate), the four oxidation steps in the cycle provide a large flow of electrons into the respiratory chain via NADH and FADH 2 and thus lead to formation of a large number of ATP molecules during oxidative phosphorylation. We saw in Chapter 14 that the energy yield from the production of two molecules of pyruvate from one molecule of glucose in glycolysis is 2 ATP and 2 NADH. In oxidative phosphorylation (Chapter 19), passage of two electrons from NADH to O 2 drives the formation of about 2.5 ATP, and passage of two electrons from FADH 2 to O 2 yields about 1.5 ATP. This stoichiometry allows us to calculate the overall yield of ATP from the complete Chapter 16 The Citric Acid Cycle614 BOX 16–2 WORKING IN BIOCHEMISTRY Citrate: A Symmetrical Molecule That Reacts Asymmetrically When compounds enriched in the heavy-carbon iso- tope 13 C and the radioactive carbon isotopes 11 C and 14 C became available about 60 years ago, they were soon put to use in tracing the pathway of carbon atoms through the citric acid cycle. One such experiment ini- tiated the controversy over the role of citrate. Acetate labeled in the carboxyl group (designated [1- 14 C] acetate) was incubated aerobically with an animal tis- sue preparation. Acetate is enzymatically converted to acetyl-CoA in animal tissues, and the pathway of the labeled carboxyl carbon of the acetyl group in the cy- cle reactions could thus be traced. H9251-Ketoglutarate was isolated from the tissue after incubation, then de- graded by known chemical reactions to establish the position(s) of the isotopic carbon. Condensation of unlabeled oxaloacetate with car- boxyl-labeled acetate would be expected to produce citrate labeled in one of the two primary carboxyl groups. Citrate is a symmetric molecule, its two ter- minal carboxyl groups being chemically indistinguish- able. Therefore, half the labeled citrate molecules were expected to yield H9251-ketoglutarate labeled in CH 3 14 COO H11002 C CH 2 O COO H11002 COO H11002 CH 2 14 COO H11002 C CH 2 HO COO H11002 COO H11002 CH 2 14 COO H11002 CH HO COO H11002 CH COO H11002 CH 14 COO H11002 CH HO COO H11002 COO H11002 H11001 Labeled acetate Oxaloacetate Labeled citrate Isocitrate CH 2 14 COO H11002 CH 2 C 14 COO H11002 CH O CH 2 COO H11002 COO H11002 Only this product was formed. This second form of labeled -ketoglutarate was also expected, but was not formed. 1 2 CO COO H11002 H9251 H9253 H9252 H9251 H9253 H9252 H9251 CH 2 FIGURE 1 Incorporation of the isotopic carbon ( 14 C) of the labeled acetyl group into H9251-ketoglutarate by the citric acid cycle. The car- bon atoms of the entering acetyl group are shown in red. 8885d_c16_601-630 1/27/04 8:54 AM Page 614 mac76 mac76:385_reb: the H9251-carboxyl group and the other half to yield H9251- keto-glutarate labeled in the H9253-carboxyl group; that is, the H9251-ketoglutarate isolated was expected to be a mix- ture of the two types of labeled molecules (Fig. 1, pathways 1 and 2 ). Contrary to this expectation, the labeled H9251-ketoglutarate isolated from the tissue suspension contained 14 C only in the H9253-carboxyl group (Fig. 1, pathway 1 ). The investigators concluded that citrate (or any other symmetric molecule) could not be an intermediate in the pathway from acetate to H9251- ketoglutarate. Rather, an asymmetric tricarboxylic acid, presumably cis-aconitate or isocitrate, must be the first product formed from condensation of acetate and oxaloacetate. In 1948, however, Alexander Ogston pointed out that although citrate has no chiral center (see Fig. 1–19), it has the potential to react asymmetrically if an enzyme with which it interacts has an active site that is asymmetric. He suggested that the active site of aconitase may have three points to which the cit- rate must be bound and that the citrate must undergo a specific three-point attachment to these binding points. As seen in Figure 2, the binding of citrate to three such points could happen in only one way, and this would account for the formation of only one type of labeled H9251-ketoglutarate. Organic molecules such as citrate that have no chiral center but are potentially capable of reacting asymmetrically with an asymmet- ric active site are now called prochiral molecules. oxidation of glucose. When both pyruvate molecules are oxidized to 6 CO 2 via the pyruvate dehydrogenase com- plex and the citric acid cycle, and the electrons are transferred to O 2 via oxidative phosphorylation, as many as 32 ATP are obtained per glucose (Table 16–1). In round numbers, this represents the conservation of 32 H11003 30.5 kJ/mol H11005 976 kJ/mol, or 34% of the theoretical maximum of about 2,840 kJ/mol available from the com- plete oxidation of glucose. These calculations employ the standard free-energy changes; when corrected for the actual free energy required to form ATP within cells (see Box 13–1), the calculated efficiency of the process is closer to 65%. Why Is the Oxidation of Acetate So Complicated? The eight-step cyclic process for oxidation of simple two- carbon acetyl groups to CO 2 may seem unnecessarily cumbersome and not in keeping with the biological prin- ciple of maximum economy. The role of the citric acid cycle is not confined to the oxidation of acetate, however. 16.2 Reactions of the Citric Acid Cycle 615 CH 2 COO H11002 HO COO H11002 (a) Susceptible bond CH 2 COO H11002 C C ZX Y (b) Z A 0 C ZX Y (c) This bond cannot be positioned correctly and is not attacked. This bond can be positioned correctly and is attacked. Active site has complementary binding points. HE Z XH11032 YH11032 ZH11032 FIGURE 2 The prochiral nature of citrate. (a) Structure of citrate; (b) schematic representation of citrate: X H11005 OOH; Y H11005 OCOO H11002 ; Z H11005 OCH 2 COO H11002 . (c) Correct complementary fit of citrate to the binding site of aconitase. There is only one way in which the three specified groups of citrate can fit on the three points of the binding site. Thus only one of the two OCH 2 COO H11002 groups is bound by aconitase. CO 2 CO 2 Acetyl-CoA Citrate Isocitrate -Ketoglutarateα Succinyl-CoA Fumarate Malate Oxaloacetate NADH NADH GTP (ATP) FADH 2 NADH Succinate Citric acid cycle FIGURE 16–13 Products of one turn of the citric acid cycle. At each turn of the cycle, three NADH, one FADH 2 , one GTP (or ATP), and two CO 2 are released in oxidative decarboxylation reactions. Here and in several following figures, all cycle reactions are shown as pro- ceeding in one direction only, but keep in mind that most of the re- actions are reversible (see Fig. 16–7). 8885d_c16_601-630 1/27/04 8:54 AM Page 615 mac76 mac76:385_reb: This pathway is the hub of intermediary metabolism. Four- and five-carbon end products of many catabolic processes feed into the cycle to serve as fuels. Oxaloac- etate and H9251-ketoglutarate, for example, are produced from aspartate and glutamate, respectively, when proteins are degraded. Under some metabolic circumstances, inter- mediates are drawn out of the cycle to be used as pre- cursors in a variety of biosynthetic pathways. The citric acid cycle, like all other metabolic path- ways, is the product of evolution, and much of this evo- lution occurred before the advent of aerobic organisms. It does not necessarily represent the shortest pathway from acetate to CO 2 , but it is the pathway that has, over time, conferred the greatest selective advantage. Early anaerobes most probably used some of the reactions of the citric acid cycle in linear biosynthetic processes. In fact, some modern anaerobic microorganisms use an in- complete citric acid cycle as a source of, not energy, but biosynthetic precursors (Fig. 16–14). These organisms use the first three reactions of the cycle to make H9251- ketoglutarate but, lacking H9251-ketoglutarate dehydroge- nase, they cannot carry out the complete set of citric acid cycle reactions. They do have the four enzymes that catalyze the reversible conversion of oxaloacetate to succinyl-CoA and can produce malate, fumarate, succi- nate, and succinyl-CoA from oxaloacetate in a reversal of the “normal” (oxidative) direction of flow through the cycle. This pathway is a fermentation, with the NADH produced by isocitrate oxidation recycled to NAD H11001 by reduction of oxaloacetate to succinate. With the evolution of cyanobacteria that produced O 2 from water, the earth’s atmosphere became aerobic and organisms were under selective pressure to develop aerobic metabolism, which, as we have seen, is much more efficient than anaerobic fermentation. Citric Acid Cycle Components Are Important Biosynthetic Intermediates In aerobic organisms, the citric acid cycle is an amphi- bolic pathway, one that serves in both catabolic and anabolic processes. Besides its role in the oxidative ca- tabolism of carbohydrates, fatty acids, and amino acids, the cycle provides precursors for many biosynthetic path- ways (Fig. 16–15), through reactions that served the same purpose in anaerobic ancestors. H9251-Ketoglutarate and oxaloacetate can, for example, serve as precursors of the amino acids aspartate and glutamate by simple transamination (Chapter 22). Through aspartate and glu- tamate, the carbons of oxaloacetate and H9251-ketoglutarate are then used to build other amino acids, as well as purine and pyrimidine nucleotides. Oxaloacetate is converted to glucose in gluconeogenesis (see Fig. 15–15). Succinyl- CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups, which serve as oxygen carriers (in hemoglobin and myoglobin) and electron carriers (in cytochromes) (see Fig. 22–23). And the cit- rate produced in some organisms is used commercially for a variety of purposes (Box 16–3). Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates As intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reactions (Fig. 16–15; Table 16–2). Under normal circumstances, the reactions by which cy- cle intermediates are siphoned off into other pathways and those by which they are replenished are in dynamic balance, so that the concentrations of the citric acid cy- cle intermediates remain almost constant. Chapter 16 The Citric Acid Cycle616 Number of ATP or reduced Number of ATP Reaction coenzyme directly formed ultimately formed* Glucose On glucose 6-phosphate H110021 ATP H110021 Fructose 6-phosphate On fructose 1,6-bisphosphate H110021 ATP H110021 2 Glyceraldehyde 3-phosphate On 2 1,3-bisphosphoglycerate H110022 NADH 3 or 5 ? 2 1,3-Bisphosphoglycerate On 2 3-phosphoglycerate H110022 ATP H110022 2 Phosphoenolpyruvate On 2 pyruvate H110022 ATP H110022 2 Pyruvate On 2 acetyl-CoA H110022 NADH H110025 2 Isocitrate On 2 H9251-ketoglutarate H110022 NADH H110025 2 H9251-Ketoglutarate On 2 succinyl-CoA H110022 NADH H110025 2 Succinyl-CoA On 2 succinate H110022 ATP (or 2 GTP) H110022 2 Succinate On 2 fumarate H110022 FADH 2 H110023 2 Malate On 2 oxaloacetate H110022 NADH H110025 Total H11002 30–32 *This is calculated as 2.5 ATP per NADH and 1.5 ATP per FADH 2 . A negative value indicates consumption. ? This number is either 3 or 5, depending on the mechanism used to shuttle NADH equivalents from the cytosol to the mitochondrial ma- trix; see Figures 19–27 and 19–28. TABLE 16–1 Stoichiometry of Coenzyme Reduction and ATP Formation in the Aerobic Oxidation of Glucose via Glycolysis, the Pyruvate Dehydrogenase Complex Reaction, the Citric Acid Cycle, and Oxidative Phosphorylation 8885d_c16_616 1/30/04 11:47 AM Page 616 mac76 mac76:385_reb: Table 16–2 shows the most common anaplerotic re- actions, all of which, in various tissues and organisms, convert either pyruvate or phosphoenolpyruvate to ox- aloacetate or malate. The most important anaplerotic re- action in mammalian liver and kidney is the reversible carboxylation of pyruvate by CO 2 to form oxaloacetate, catalyzed by pyruvate carboxylase. When the citric acid cycle is deficient in oxaloacetate or any other intermedi- ates, pyruvate is carboxylated to produce more oxalo- acetate. The enzymatic addition of a carboxyl group to pyruvate requires energy, which is supplied by ATP—the free energy required to attach a carboxyl group to pyru- vate is about equal to the free energy available from ATP. Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its pos- itive allosteric modulator. Whenever acetyl-CoA, the fuel for the citric acid cycle, is present in excess, it stimu- lates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction. The other anaplerotic reactions shown in Table 16–2 are also regulated to keep the level of intermediates high enough to support the activity of the citric acid cycle. Phosphoenolpyruvate (PEP) carboxylase, for example, is activated by the glycolytic intermediate fructose 1,6- bisphosphate, which accumulates when the citric acid cycle operates too slowly to process the pyruvate gen- erated by glycolysis. 16.2 Reactions of the Citric Acid Cycle 617 -Ketoglutarateα Biosynthetic products (amino acids, nucleotides, heme, etc.) Succinyl-CoA PEP or pyruvate CO 2 Acetyl-CoA Citrate Oxaloacetate Malate Fumarate Isocitrate Succinate FIGURE 16–14 Biosynthetic precursors produced by an incomplete citric acid cycle in anaerobic bacteria. These anaerobes lack H9251- ketoglutarate dehydrogenase and therefore cannot carry out the complete citric acid cycle. H9251-Ketoglutarate and succinyl-CoA serve as precursors in a variety of biosynthetic pathways. (See Fig. 16–13 for the “normal” direction of these reactions in the citric acid cycle.) Phosphoenolpyruvate (PEP) PEP carboxylase PEP carboxykinase pyruvate carboxylase Porphyrins, heme Glutamate Purines Arginine Proline Glutamine Pyrimidines Aspartate Asparagine Serine Glycine Cysteine Phenylalanine Tyrosine Tryptophan Glucose Fatty acids, sterols Pyruvate malic enzyme Pyruvate -KetoglutarateαMalate Succinyl-CoA Oxaloacetate Acetyl-CoA Citrate Citric acid cycle FIGURE 16–15 Role of the citric acid cycle in anabolism. Intermediates of the citric acid cycle are drawn off as precursors in many biosynthetic pathways. Shown in red are four anaplerotic reactions that replenish depleted cycle intermediates (see Table 16–2). 8885d_c16_601-630 1/27/04 8:54 AM Page 617 mac76 mac76:385_reb: Biotin in Pyruvate Carboxylase Carries CO 2 Groups The pyruvate carboxylase reaction requires the vitamin biotin (Fig. 16–16), which is the prosthetic group of the enzyme. Biotin plays a key role in many carboxyla- tion reactions. It is a specialized carrier of one-carbon groups in their most oxidized form: CO 2 . (The transfer of one-carbon groups in more reduced forms is medi- ated by other cofactors, notably tetrahydrofolate and S-adenosylmethionine, as described in Chapter 18.) Carboxyl groups are activated in a reaction that splits ATP and joins CO 2 to enzyme-bound biotin. This “acti- vated” CO 2 is then passed to an acceptor (pyruvate in this case) in a carboxylation reaction. Pyruvate carboxylase has four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage to the ε-amino group of a spe- cific Lys residue in the enzyme active site. Carboxylation of pyruvate proceeds in two steps (Fig. 16–16): first, a carboxyl group derived from HCO 3 H11002 is attached to biotin, Chapter 16 The Citric Acid Cycle618 TABLE 16–2 Anaplerotic Reactions Reaction Tissue(s)/organism(s) Pyruvate H11001 HCO 3 H11002 H11001 ATP oxaloacetate H11001 ADP H11001 P i Liver, kidney Phosphoenolpyruvate H11001 CO 2 H11001 GDP oxaloacetate H11001 GTP Heart, skeletal muscle Phosphoenolpyruvate H11001 HCO 3 H11002 oxaloacetate H11001 P i Higher plants, yeast, bacteria Pyruvate H11001 HCO 3 H11002 H11001 NAD(P)H malate H11001 NAD(P) H11001 Widely distributed in eukaryotes and prokaryotes pyruvate carboxylase PEP carboxykinase PEP carboxylase malic enzyme 888888888888z y888888888888 888888888888z y888888888888 888888888888z y888888888888 888888888888z y888888888888 BOX 16–3 THE WORLD OF BIOCHEMISTRY Citrate Synthase, Soda Pop, and the World Food Supply Citrate has a number of important industrial applica- tions. A quick examination of the ingredients in most soft drinks reveals the common use of citric acid to provide a tart or fruity flavor. Citric acid is also used as a plasticizer and foam inhibitor in the manufacture of certain resins, as a mordant to brighten colors, and as an antioxidant to preserve the flavors of foods. Citric acid is produced industrially by growing the fungus Aspergillus niger in the presence of an inex- pensive sugar source, usually beet molasses. Culture conditions are designed to inhibit the reactions of the citric acid cycle such that citrate accumulates. On a grander scale, citric acid may one day play a spectacular role in the alleviation of world hunger. With its three negatively charged carboxyl groups, cit- rate is a good chelator of metal ions, and some plants exploit this property by releasing citrate into the soil, where it binds metal ions and prevents their absorp- tion by the plant. Of particular importance is the alu- minum ion (Al 3H11001 ), which is toxic to many plants and causes decreased crop yields on 30% to 40% of the world’s arable land. Aluminum is the most abundant metal in the earth’s crust, yet it occurs mostly in chem- ical compounds, such as Al(OH) 3 , that are biologically inert. However, when soil pH is less than 5, Al 3H11001 be- comes soluble and thus can be absorbed by plant roots. Acidic soil and Al 3H11001 toxicity are most prevalent in the tropics, where maize yields can be depressed by as much as 80%. In Mexico, Al 3H11001 toxicity limits pa- paya production to 20,000 hectares, instead of the 3 million hectares that could theoretically be cultivated. One solution would be to raise soil pH with lime, but this is economically and environmentally unsound. An alternative would be to breed Al 3H11001 -resistant plants. Naturally resistant plants do exist, and these provide the means for a third solution: transferring resistance to crop plants by genetic engineering. A group of researchers in Mexico has genetically engineered tobacco and papaya plants to express el- evated levels of bacterial citrate synthase. These plants secrete five to six times their normal amount of Al 3H11001 -chelating citric acid and can grow in soils with Al 3H11001 levels ten times those at which control plants can grow. This degree of resistance would allow Mexico to grow papaya on the 3 million hectares of land cur- rently rendered unsuitable by Al 3H11001 . Given projected levels of population growth, world food production must more than triple in the next 50 years to adequately feed 9.6 billion people. A long-term solution may turn on increasing crop productivity on the arable land affected by aluminum toxicity, and citric acid may play an important role in achieving this goal. 8885d_c16_601-630 1/27/04 8:54 AM Page 618 mac76 mac76:385_reb: 16.2 Reactions of the Citric Acid Cycle 619 : HN NH O S Catalytic site 1 Catalytic site 2 1 O – O P P O O – – O Transfer of carboxybiotin (activated CO 2 ) to second catalytic site NH Lys O P ATP ADP Carboxyphosphate 2 3 P i 5 C Bicar- bonate Biotinyl- lysine Pyruvate carboxylase HO O H C CH 2 C O O – O OO – C C CH 2 C O O – O HN NH O S NNH O S NH H + O C – O O NH O C O O C O O O – O P O – OC O O H Carboxybiotinyl-enzyme 4 NHN O S NH O C O – O Pyruvate Oxaloacetate 6 7 NHN O – S NH O C CH 2 C O – O – O C O O NH Pyruvate enolate HN O S NH O AdenineRib MECHANISM FIGURE 16–16 The role of biotin in the reaction cat- alyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the H9280-amino group of a Lys residue, form- ing biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed by separate active sites on the en- zyme as exemplified by the pyruvate carboxylase reaction. In the first phase (steps H220711toH220713 ), bicarbonate is converted to the more acti- vated CO 2 , and then used to carboxylate biotin. The bicarbonate is first activated by reaction with ATP to form carboxyphosphate (step H220711 ), which breaks down to carbon dioxide (step H220712 ). In effect, the bicarbonate is dehydrated by its reaction with ATP, and the CO 2 can react with biotin to form carboxybiotin (step H220713 ). The biotin acts as a carrier to transport the CO 2 from one active site to another on the same enzyme (step H220714 ). In the second phase of the reaction (steps H220715toH220717 ), catalyzed in a second active site, the CO 2 reacts with pyruvate to form oxaloacetate. The CO 2 is released in the second ac- tive site (step H220715 ). Pyruvate is converted to its enolate form in step H220716 , transferring a proton to biotin. The enolate then attacks the CO 2 to generate oxaloacetate in the final step of the reaction (step H220717). 8885d_c16_601-630 1/27/04 8:54 AM Page 619 mac76 mac76:385_reb: Chapter 16 The Citric Acid Cycle620 S S S O O N N N H N H CH N H NHHN HS (E 2 ) CH 3 CH 3 Dihydrolipoyl transacetylase C O O O CH N H OH O H11002 C O O O O Ser Acyl carrier protein P O Lipoate Biotin Pantothenate H9252-Mercapto- ethylamine Lys residue Pyruvate carboxylase FIGURE 16–17 Biological tethers. The cofactors lipoate, biotin, and the combination of H9252-mercaptoethylamine and pantothenate form long, flexible arms in the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next. The group shaded pink is in each case the point of attachment of the activated intermediate to the tether. then the carboxyl group is transferred to pyruvate to form oxaloacetate. These two steps occur at separate active sites; the long flexible arm of biotin transfers ac- tivated carboxyl groups from the first active site to the second, functioning much like the long lipoyllysine arm of E 2 in the PDH complex (Fig. 16–6) and the long arm of the CoA-like moiety in the acyl carrier protein involved in fatty acid synthesis (see Fig. 21–5); these are com- pared in Figure 16–17. Lipoate, biotin, and pantothen- ate all enter cells on the same transporter; all become covalently attached to proteins by similar reactions; and all provide a flexible tether that allows bound reaction intermediates to move from one active site to another in an enzyme complex, without dissociating from it—all, that is, participate in substrate channeling. Biotin is a vitamin required in the human diet; it is abundant in many foods and is synthesized by intestinal bacteria. Biotin deficiency is rare, but can sometimes be caused by a diet rich in raw eggs. Egg whites contain a large amount of the protein avidin (M r 70,000), which binds very tightly to biotin and prevents its absorption in the intestine. The avidin of egg whites may be a de- fense mechanism for the potential chick embryo, in- hibiting the growth of bacteria. When eggs are cooked, avidin is denatured (and thereby inactivated) along with all other egg white proteins. Purified avidin is a useful reagent in biochemistry and cell biology. A protein that contains covalently bound biotin (derived experimen- tally or produced in vivo) can be recovered by affinity chromatography (see Fig. 3–18c) based on biotin’s strong affinity for avidin. The protein is then eluted from the column with an excess of free biotin. The very high affinity of biotin for avidin is also used in the laboratory in the form of a molecular glue that can hold two struc- tures together (see Fig. 19–25). SUMMARY 16.2 Reactions of the Citric Acid Cycle ■ The citric acid cycle (Krebs cycle, TCA cycle) is a nearly universal central catabolic pathway in which compounds derived from the break- down of carbohydrates, fats, and proteins are oxidized to CO 2 , with most of the energy of oxidation temporarily held in the electron carriers FADH 2 and NADH. During aerobic metabolism, these electrons are transferred to O 2 and the energy of electron flow is trapped as ATP. ■ Acetyl-CoA enters the citric acid cycle (in the mitochondria of eukaryotes, the cytosol of prokaryotes) as citrate synthase catalyzes its condensation with oxaloacetate to form citrate. ■ In seven sequential reactions, including two decarboxylations, the citric acid cycle converts citrate to oxaloacetate and releases two CO 2 . The pathway is cyclic in that the intermediates of the cycle are not used up; for each oxalo- acetate consumed in the path, one is produced. ■ For each acetyl-CoA oxidized by the citric acid cycle, the energy gain consists of three molecules of NADH, one FADH 2 , and one nucleoside triphosphate (either ATP or GTP). ■ Besides acetyl-CoA, any compound that gives rise to a four- or five-carbon intermediate of the citric acid cycle—for example, the break- down products of many amino acids—can be oxidized by the cycle. ■ The citric acid cycle is amphibolic, serving in both catabolism and anabolism; cycle inter- mediates can be drawn off and used as the starting material for a variety of biosynthetic products. ■ When intermediates are shunted from the citric acid cycle to other pathways, they are replenished by several anaplerotic reactions, which produce four-carbon intermediates by carboxylation of three-carbon compounds; these reactions are catalyzed by pyruvate carboxylase, PEP carboxykinase, PEP carboxylase, and malic enzyme. Enzymes that catalyze carboxylations commonly employ biotin to activate CO 2 and to carry it to acceptors such as pyruvate or phosphoenolpyruvate. 8885d_c16_601-630 1/27/04 8:54 AM Page 620 mac76 mac76:385_reb: NADH FADH 2 Acetyl-CoA Citrate Isocitrate -Ketoglutarateα H9251 Succinyl-CoA Malate Oxaloacetate Pyruvate pyruvate dehydrogenase complex ATP, acetyl-CoA, NADH, fatty acids AMP, CoA, NAD H11001 , Ca 2H11001 citrate synthase NADH, succinyl-CoA, citrate, ATP ADP isocitrate dehydrogenase ATP Ca 2H11001 , ADP succinyl-CoA, NADH Ca 2H11001 -ketoglutarate dehydrogenase complex succinate dehydrogenase malate dehydrogenase GTP (ATP) Citric acid cycle 16.3 Regulation of the Citric Acid Cycle As we have seen in Chapter 15, the regulation of key enzymes in metabolic pathways, by allosteric effectors and by covalent modification, ensures the production of intermediates at the rates required to keep the cell in a stable steady state while avoiding wasteful overproduc- tion. The flow of carbon atoms from pyruvate into and through the citric acid cycle is under tight regulation at two levels: the conversion of pyruvate to acetyl-CoA, the starting material for the cycle (the pyruvate dehydro- genase complex reaction), and the entry of acetyl-CoA into the cycle (the citrate synthase reaction). Acetyl- CoA is also produced by pathways other than the PDH complex reaction—most cells produce acetyl-CoA from the oxidation of fatty acids and certain amino acids— and the availability of intermediates from these other pathways is important in the regulation of pyruvate oxi- dation and of the citric acid cycle. The cycle is also regu- lated at the isocitrate dehydrogenase and H9251-ketoglutarate dehydrogenase reactions. Production of Acetyl-CoA by the Pyruvate Dehydrogenase Complex Is Regulated by Allosteric and Covalent Mechanisms The PDH complex of mammals is strongly inhibited by ATP and by acetyl-CoA and NADH, the products of the reaction catalyzed by the complex (Fig. 16–18). The al- losteric inhibition of pyruvate oxidation is greatly en- hanced when long-chain fatty acids are available. AMP, CoA, and NAD H11001 , all of which accumulate when too lit- tle acetate flows into the citric acid cycle, allosterically activate the PDH complex. Thus, this enzyme activity is turned off when ample fuel is available in the form of fatty acids and acetyl-CoA and when the cell’s [ATP]/[ADP] and [NADH]/[NAD H11001 ] ratios are high, and it is turned on again when energy demands are high and the cell requires greater flux of acetyl-CoA into the cit- ric acid cycle. In mammals, these allosteric regulatory mechanisms are complemented by a second level of regulation: co- valent protein modification. The PDH complex is inhib- ited by reversible phosphorylation of a specific Ser residue on one of the two subunits of E 1 . As noted ear- lier, in addition to the enzymes E 1 , E 2 , and E 3 , the mam- malian PDH complex contains two regulatory proteins whose sole purpose is to regulate the activity of the complex. A specific protein kinase phosphorylates and thereby inactivates E 1 , and a specific phosphoprotein phosphatase removes the phosphoryl group by hydrol- ysis and thereby activates E 1 . The kinase is allosterically activated by ATP: when [ATP] is high (reflecting a suf- ficient supply of energy), the PDH complex is inactivated by phosphorylation of E 1 . When [ATP] declines, kinase activity decreases and phosphatase action removes the phosphoryl groups from E 1 , activating the complex. The PDH complex of plants, located in the mito- chondrial matrix and in plastids, is inhibited by its prod- ucts, NADH and acetyl-CoA. The plant mitochondrial 16.3 Regulation of the Citric Acid Cycle 621 FIGURE 16–18 Regulation of metabolite flow from the PDH complex through the citric acid cycle. The PDH complex is allosterically inhibited when [ATP]/[ADP], [NADH]/[NAD H11001 ], and [acetyl-CoA]/[CoA] ratios are high, indicating an energy-sufficient metabolic state. When these ratios decrease, allosteric activation of pyruvate oxidation results. The rate of flow through the citric acid cycle can be limited by the availability of the citrate synthase substrates, oxaloacetate and acetyl-CoA, or of NAD H11001 , which is depleted by its conversion to NADH, slowing the three NAD-dependent oxidation steps. Feedback inhibition by succinyl-CoA, citrate, and ATP also slows the cycle by inhibiting early steps. In muscle tissue, Ca 2H11001 signals contraction and, as shown here, stimulates energy-yielding metabolism to replace the ATP consumed by contraction. 8885d_c16_601-630 1/27/04 8:54 AM Page 621 mac76 mac76:385_reb: enzyme is also regulated by reversible phosphorylation; pyruvate inhibits the kinase, thus activating the PDH complex, and NH 4 H11001 stimulates the kinase, causing inac- tivation of the complex. The PDH complex of E. coli is under allosteric regulation similar to that of the mam- malian enzyme, but it does not seem to be regulated by phosphorylation. The Citric Acid Cycle Is Regulated at Its Three Exergonic Steps The flow of metabolites through the citric acid cycle is under stringent regulation. Three factors govern the rate of flux through the cycle: substrate availability, in- hibition by accumulating products, and allosteric feed- back inhibition of the enzymes that catalyze early steps in the cycle. Each of the three strongly exergonic steps in the cycle—those catalyzed by citrate synthase, isocitrate dehydrogenase, and H9251-ketoglutarate dehydrogenase (Fig. 16–18)—can become the rate-limiting step under some circumstances. The availability of the substrates for citrate synthase (acetyl-CoA and oxaloacetate) varies with the metabolic state of the cell and sometimes limits the rate of citrate formation. NADH, a product of isocitrate and H9251-ketoglutarate oxidation, accumulates under some conditions, and at high [NADH]/[NAD H11001 ] both dehydrogenase reactions are severely inhibited by mass action. Similarly, in the cell, the malate dehy- drogenase reaction is essentially at equilibrium (that is, it is substrate-limited, and when [NADH]/[NAD H11001 ] is high the concentration of oxaloacetate is low, slowing the first step in the cycle. Product accumulation inhibits all three limiting steps of the cycle: succinyl-CoA inhibits H9251- ketoglutarate dehydrogenase (and also citrate synthase); citrate blocks citrate synthase; and the end product, ATP, inhibits both citrate synthase and isocitrate dehy- drogenase. The inhibition of citrate synthase by ATP is relieved by ADP, an allosteric activator of this enzyme. In vertebrate muscle, Ca 2H11001 , the signal for contraction and for a concomitant increase in demand for ATP, acti- vates both isocitrate dehydrogenase and H9251-ketoglutarate dehydrogenase, as well as the PDH complex. In short, the concentrations of substrates and intermediates in the citric acid cycle set the flux through this pathway at a rate that provides optimal concentrations of ATP and NADH. Under normal conditions, the rates of glycolysis and of the citric acid cycle are integrated so that only as much glucose is metabolized to pyruvate as is needed to supply the citric acid cycle with its fuel, the acetyl groups of acetyl-CoA. Pyruvate, lactate, and acetyl-CoA are normally maintained at steady-state concentrations. The rate of glycolysis is matched to the rate of the cit- ric acid cycle not only through its inhibition by high lev- els of ATP and NADH, which are common to both the Chapter 16 The Citric Acid Cycle622 glycolytic and respiratory stages of glucose oxidation, but also by the concentration of citrate. Citrate, the product of the first step of the citric acid cycle, is an important allosteric inhibitor of phosphofructokinase-1 in the glycolytic pathway (see Fig. 15–18). Substrate Channeling through Multienzyme Complexes May Occur in the Citric Acid Cycle Although the enzymes of the citric acid cycle are usu- ally described as soluble components of the mitochon- drial matrix (except for succinate dehydrogenase, which is membrane-bound), growing evidence suggests that within the mitochondrion these enzymes exist as multi- enzyme complexes. The classic approach of enzymol- ogy—purification of individual proteins from extracts of broken cells—was applied with great success to the cit- ric acid cycle enzymes. However, the first casualty of cell breakage is higher-level organization within the cell—the noncovalent, reversible interaction of one protein with another, or of an enzyme with some structural compo- nent such as a membrane, microtubule, or microfilament. When cells are broken open, their contents, including enzymes, are diluted 100- or 1,000-fold (Fig. 16–19). Several types of evidence suggest that, in cells, multi- enzyme complexes ensure efficient passage of the prod- uct of one enzyme reaction to the next enzyme in the pathway. Such complexes are called metabolons. Cer- tain enzymes of the citric acid cycle have been isolated together as supramolecular aggregates, or have been found associated with the inner mitochondrial mem- brane, or have been shown to diffuse in the mitochon- drial matrix more slowly than expected for the individ- ual protein in solution. There is strong evidence for substrate channeling through multienzyme complexes in In the cytosol, high concentrations of enzymes 1, 2, and 3 favor their association. In extract of broken cells, dilution by buffer reduces the concentrations of enzymes 1, 2, and 3, favoring their dissociation. FIGURE 16–19 Dilution of a solution containing a noncovalent pro- tein complex—such as one consisting of three enzymes—favors dis- sociation of the complex into its constituents. 8885d_c16_622 1/30/04 11:47 AM Page 622 mac76 mac76:385_reb: other metabolic pathways, and many enzymes thought of as “soluble” probably function in the cell as highly or- ganized complexes that channel intermediates. We will encounter other examples of channeling when we dis- cuss the biosynthesis of amino acids and nucleotides in Chapter 22. SUMMARY 16.3 Regulation of the Citric Acid Cycle ■ The overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate dehydrogenase, and H9251-ketoglutarate dehydrogenase. These fluxes are largely determined by the concentrations of substrates and products: the end products ATP and NADH are inhibitory, and the substrates NAD H11001 and ADP are stimulatory. ■ The production of acetyl-CoA for the citric acid cycle by the PDH complex is inhibited allosterically by metabolites that signal a sufficiency of metabolic energy (ATP, acetyl- CoA, NADH, and fatty acids) and stimulated by metabolites that indicate a reduced energy supply (AMP, NAD H11001 , CoA). 16.4 The Glyoxylate Cycle Vertebrates cannot convert fatty acids, or the acetate derived from them, to carbohydrates. Conversion of phosphoenolpyruvate to pyruvate (p. 532) and of pyru- vate to acetyl-CoA (Fig. 16–2) are so exergonic as to be essentially irreversible. If a cell cannot convert acetate into phosphoenolpyruvate, acetate cannot serve as the starting material for the gluconeogenic pathway, which leads from phosphoenolpyruvate to glucose (see Fig. 15–15). Without this capacity, then, a cell or organism is unable to convert fuels or metabolites that are de- graded to acetate (fatty acids and certain amino acids) into carbohydrates. As noted in the discussion of anaplerotic reactions (Table 16–2), phosphoenolpyruvate can be synthesized from oxaloacetate in the reversible reaction catalyzed by PEP carboxykinase: Oxaloacetate H11001 GTP 34 phosphoenolpyruvate H11001 CO 2 H11001 GDP Because the carbon atoms of acetate molecules that enter the citric acid cycle appear eight steps later in oxaloacetate, it might seem that this pathway could gen- erate oxaloacetate from acetate and thus generate phosphoenolpyruvate for gluconeogenesis. However, as an examination of the stoichiometry of the citric acid cycle shows, there is no net conversion of acetate to ox- aloacetate; in vertebrates, for every two carbons that enter the cycle as acetyl-CoA, two leave as CO 2 . In many organisms other than vertebrates, the glyoxylate cycle serves as a mechanism for converting acetate to carbohydrate. The Glyoxylate Cycle Produces Four-Carbon Compounds from Acetate In plants, certain invertebrates, and some microorgan- isms (including E. coli and yeast) acetate can serve both as an energy-rich fuel and as a source of phospho- enolpyruvate for carbohydrate synthesis. In these or- ganisms, enzymes of the glyoxylate cycle catalyze the net conversion of acetate to succinate or other four- carbon intermediates of the citric acid cycle: 2 Acetyl-CoA H11001 NAD H11001 H11001 2H 2 O On succinate H11001 2CoA H11001 NADH H11001 H H11001 In the glyoxylate cycle, acetyl-CoA condenses with ox- aloacetate to form citrate, and citrate is converted to isocitrate, exactly as in the citric acid cycle. The next step, however, is not the breakdown of isocitrate by iso- citrate dehydrogenase but the cleavage of isocitrate by isocitrate lyase, forming succinate and glyoxylate. The glyoxylate then condenses with a second molecule of acetyl-CoA to yield malate, in a reaction catalyzed by malate synthase. The malate is subsequently oxidized to oxaloacetate, which can condense with another mol- ecule of acetyl-CoA to start another turn of the cycle (Fig. 16–20). Each turn of the glyoxylate cycle con- sumes two molecules of acetyl-CoA and produces one molecule of succinate, which is then available for bio- synthetic purposes. The succinate may be converted through fumarate and malate into oxaloacetate, which can then be converted to phosphoenolpyruvate by PEP carboxykinase, and thus to glucose by gluconeogenesis. Vertebrates do not have the enzymes specific to the gly- oxylate cycle (isocitrate lyase and malate synthase) and therefore cannot bring about the net synthesis of glu- cose from lipids. In plants, the enzymes of the glyoxylate cycle are sequestered in membrane-bounded organelles called glyoxysomes, which are specialized peroxisomes (Fig. 16–21). Those enzymes common to the citric acid and glyoxylate cycles have two isozymes, one specific to mitochondria, the other to glyoxysomes. Glyoxysomes are not present in all plant tissues at all times. They de- velop in lipid-rich seeds during germination, before the developing plant acquires the ability to make glucose by photosynthesis. In addition to glyoxylate cycle enzymes, glyoxysomes contain all the enzymes needed for the degradation of the fatty acids stored in seed oils (see Fig. 17–13). Acetyl-CoA formed from lipid breakdown is converted to succinate via the glyoxylate cycle, and the succinate is exported to mitochondria, where citric 16.4 The Glyoxylate Cycle 623 8885d_c16_601-630 1/27/04 8:54 AM Page 623 mac76 mac76:385_reb: acid cycle enzymes transform it to malate. A cytosolic isozyme of malate dehydrogenase oxidizes malate to ox- aloacetate, a precursor for gluconeogenesis. Germinat- ing seeds can therefore convert the carbon of stored lipids into glucose. The Citric Acid and Glyoxylate Cycles Are Coordinately Regulated In germinating seeds, the enzymatic transformations of dicarboxylic and tricarboxylic acids occur in three in- tracellular compartments: mitochondria, glyoxysomes, and the cytosol. There is a continuous interchange of metabolites among these compartments (Fig. 16–22). The carbon skeleton of oxaloacetate from the citric acid cycle (in the mitochondrion) is carried to the gly- oxysome in the form of aspartate. Aspartate is converted Chapter 16 The Citric Acid Cycle624 C CH 2 NADH NAD H11001 O COO H11002 COO H11002 C CH 2 HO COO H11002 CH 2 COO H11002 COO H11002 Citrate CH CH 2 COO H11002 COO H11002 CH 2 COO H11002 CH 2 COO H11002 CH COO H11002 HO C CO O H O H11002 CH 2 COO H11002 COO H11002 CHHO Isocitrate Succinate Oxaloacetate CH 3 O CS - CoA Acetyl - CoA Acetyl - CoA CH 3 O CS - CoA Malate Glyoxylate citrate synthase isocitrate lyase malate synthase malate dehydrogenase aconitase Glyoxylate cycle FIGURE 16–20 Glyoxylate cycle. The citrate synthase, aconitase, and malate dehydrogenase of the glyoxylate cycle are isozymes of the cit- ric acid cycle enzymes; isocitrate lyase and malate synthase are unique to the glyoxylate cycle. Notice that two acetyl groups (pink) enter the cycle and four carbons leave as succinate (blue). The glyoxylate cy- cle was elucidated by Hans Kornberg and Neil Madsen in the labo- ratory of Hans Krebs. Lipid body Glyoxysome Mitochondria FIGURE 16–21 Electron micrograph of a germinating cucumber seed, showing a glyoxysome, mitochondria, and surrounding lipid bodies. to oxaloacetate, which condenses with acetyl-CoA de- rived from fatty acid breakdown. The citrate thus formed is converted to isocitrate by aconitase, then split into glyoxylate and succinate by isocitrate lyase. The succinate returns to the mitochondrion, where it reen- ters the citric acid cycle and is transformed into malate, which enters the cytosol and is oxidized (by cytosolic malate dehydrogenase) to oxaloacetate. Oxaloacetate is converted via gluconeogenesis into hexoses and su- crose, which can be transported to the growing roots and shoot. Four distinct pathways participate in these conversions: fatty acid breakdown to acetyl-CoA (in gly- oxysomes), the glyoxylate cycle (in glyoxysomes), the citric acid cycle (in mitochondria), and gluconeogene- sis (in the cytosol). The sharing of common intermediates requires that these pathways be coordinately regulated. Isocitrate is a crucial intermediate, at the branch point between the glyoxylate and citric acid cycles (Fig. 16–23). Isocitrate dehydrogenase is regulated by covalent modification: a specific protein kinase phosphorylates and thereby in- activates the dehydrogenase. This inactivation shunts isocitrate to the glyoxylate cycle, where it begins the synthetic route toward glucose. A phosphoprotein phos- phatase removes the phosphoryl group from isocitrate dehydrogenase, reactivating the enzyme and sending more isocitrate through the energy-yielding citric acid cycle. The regulatory protein kinase and phosphopro- tein phosphatase are separate enzymatic activities of a single polypeptide. Some bacteria, including E. coli, have the full com- plement of enzymes for the glyoxylate and citric acid cycles in the cytosol and can therefore grow on acetate as their sole source of carbon and energy. The phospho- protein phosphatase that activates isocitrate dehydroge- nase is stimulated by intermediates of the citric acid cycle and glycolysis and by indicators of reduced cellu- lar energy supply (Fig. 16–23). The same metabolites inhibit the protein kinase activity of the bifunctional polypeptide. Thus, the accumulation of intermediates of 8885d_c16_601-630 1/27/04 8:54 AM Page 624 mac76 mac76:385_reb: the central energy-yielding pathways—indicating en- ergy depletion—results in the activation of isocitrate de- hydrogenase. When the concentration of these regula- tors falls, signaling a sufficient flux through the energy-yielding citric acid cycle, isocitrate dehydroge- nase is inactivated by the protein kinase. 16.4 The Glyoxylate Cycle 625 Mitochondrion Hexoses Acetyl-CoA Fatty acids Triacylglycerols Lipid body gluconeogenesisGlyoxysome Acetyl-CoA Fatty acids Malate Oxaloacetate Sucrose Succinate Cytosol Isocitrate Citrate Fumarate Malate Citric acid cycle Oxaloacetate Succinate Oxaloacetate Malate Glyoxylate cycle Glyoxylate Citrate FIGURE 16–22 Relationship between the glyoxylate and citric acid cycles. The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultaneously with, and mesh with, those of the citric acid cycle (in mitochondria), as intermediates pass between these compartments. The conversion of succinate to oxaloacetate is catalyzed by citric acid cycle enzymes. The oxidation of fatty acids to acetyl-CoA is described in Chapter 17; the synthesis of hexoses from oxaloacetate is described in Chapter 20. The same intermediates of glycolysis and the citric acid cycle that activate isocitrate dehydrogenase are allosteric inhibitors of isocitrate lyase. When energy- yielding metabolism is sufficiently fast to keep the concentrations of glycolytic and citric acid cycle inter- mediates low, isocitrate dehydrogenase is inactivated, the inhibition of isocitrate lyase is relieved, and isocitrate flows into the glyoxylate pathway, to be used in the biosynthesis of carbohydrates, amino acids, and other cellular components. ATP Acetyl-CoA Isocitrate NADH, FADH 2 oxidative phosphorylation Amino acids, nucleotides Oxaloacetate gluconeogenesis Glucose protein kinase isocitrate dehydrogenase isocitrate lyase phosphatase intermediates of citric acid cycle and glycolysis, AMP, ADP intermediates of citric acid cycle and glycolysis, AMP, ADP -Ketoglutarateα Succinate, glyoxylate Citric acid cycle Glyoxylate cycle FIGURE 16–23 Coordinated regulation of glyoxylate and citric acid cycles. Regulation of isocitrate dehydrogenase activity determines the partitioning of isocitrate between the glyoxylate and citric acid cycles. When the enzyme is inactivated by phosphorylation (by a specific pro- tein kinase), isocitrate is directed into biosynthetic reactions via the glyoxylate cycle. When the enzyme is activated by dephosphorylation (by a specific phosphatase), isocitrate enters the citric acid cycle and ATP is produced. 8885d_c16_625 1/30/04 11:48 AM Page 625 mac76 mac76:385_reb: Key Terms respiration 601 cellular respiration 601 citric acid cycle 601 tricarboxylic acid (TCA) cycle 601 Krebs cycle 601 pyruvate dehydrogenase (PDH) complex 602 oxidative decarboxylation 602 thioester 603 lipoate 603 substrate channeling 605 iron-sulfur center 609 H9251-ketoglutarate dehydrogenase complex 610 nucleoside diphosphate kinase 612 synthases 613 synthetases 613 ligases 613 lyases 613 kinases 613 phosphorylases 613 phosphatases 613 prochiral molecule 615 amphibolic pathway 616 anaplerotic reaction 616 biotin 618 avidin 620 metabolon 622 glyoxylate cycle 623 Terms in bold are defined in the glossary. Further Reading General Holmes, F.L. (1990, 1993) Hans Krebs, Vol 1: Formation of a Scientific Life, 1900–1933; Vol. 2: Architect of Intermediary Metabolism, 1933–1937, Oxford University Press, Oxford. A scientific and personal biography of Krebs by an eminent his- torian of science, with a thorough description of the work that revealed the urea and citric acid cycles. Kay, J. & Weitzman, P.D.J. (eds) (1987) Krebs’ Citric Acid Cycle: Half a Century and Still Turning, Biochemical Society Symposium 54, The Biochemical Society, London. A multiauthor book on the citric acid cycle, including molecular genetics, regulatory mechanisms, variations on the cycle in microorganisms from unusual ecological niches, and evolution of the pathway. Especially relevant are the chapters by H. Gest (Evolutionary Roots of the Citric Acid Cycle in Prokaryotes), W. H. Holms (Control of Flux through the Citric Acid Cycle and the Glyoxylate Bypass in Escherichia coli), and R. N. Perham et al. (H9251-Keto Acid Dehydrogenase Complexes). Pyruvate Dehydrogenase Complex Harris, R.A., Bowker-Kinley, M.M., Huang, B., & Wu, P. (2002) Regulation of the activity of the pyruvate dehydrogenase complex. Adv. Enzyme Regul. 42, 249–259. Milne, J.L.S., Shi, D., Rosenthal, P.B., Sunshine, J.S., Domingo, G.J., Wu, X., Brooks, B.R., Perham, R.N., Hender- son, R., & Subramaniam, S. (2002) Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine. EMBO J. 21, 5587–5598. Beautiful illustration of the power of image reconstruction methodology with cryoelectron microscopy, here used to develop a plausible model for the structure of the PDH com- plex. Compare this model with that in the paper by Zhou et al. Perham, R.N. (2000) Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions. Annu. Rev. Biochem. 69, 961–1004. Review of the roles of swinging arms containing lipoate, biotin, and pantothenate in substrate channeling through multienzyme complexes. Zhou, Z.H., McCarthy, D.B., O’Conner, C.M., Reed, L.J., & Stoops, J.K. (2001) The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proc. Natl. Acad. Sci. USA 98, 14,802–14,807. Another striking paper in which image reconstruction with cryoelectron microscopy yields a model of the PDH complex. Compare this model with that in the paper by Milne et al. Citric Acid Cycle Enzymes Fraser, M.D., James, M.N., Bridger, W.A., & Wolodko, W.T. (1999) A detailed structural description of Escherichia coli succinyl-CoA synthetase. J. Mol. Biol. 285, 1633–1653. (See also the erratum in J. Mol. Biol. 288, 501 (1999).) SUMMARY 16.4 The Glyoxylate Cycle ■ The glyoxylate cycle is active in the germinating seeds of some plants and in certain microorganisms that can live on acetate as the sole carbon source. In plants, the pathway takes place in glyoxysomes in seedlings. It involves several citric acid cycle enzymes and two additional enzymes: isocitrate lyase and malate synthase. ■ In the glyoxylate cycle, the bypassing of the two decarboxylation steps of the citric acid Chapter 16 The Citric Acid Cycle626 cycle makes possible the net formation of succinate, oxaloacetate, and other cycle intermediates from acetyl-CoA. Oxaloacetate thus formed can be used to synthesize glucose via gluconeogenesis. ■ The partitioning of isocitrate between the citric acid cycle and the glyoxylate cycle is controlled at the level of isocitrate dehydrogenase, which is regulated by reversible phosphorylation. ■ Vertebrates lack the glyoxylate cycle and cannot synthesize glucose from acetate or the fatty acids that give rise to acetyl-CoA. 8885d_c16_601-630 1/27/04 8:54 AM Page 626 mac76 mac76:385_reb: Chapter 16 Problems 627 Goward, C.R. & Nicholls, D.J. (1994) Malate dehydrogenase: a model for structure, evolution, and catalysis. Protein Sci. 3, 1883–1888. A good, short review. Hagerhall, C. (1997) Succinate:quinone oxidoreductases: variations on a conserved theme. Biochim. Biophys. Acta 1320, 107–141. A review of the structure and function of succinate dehydrogenases. Jitrapakdee, S. & Wallace, J.C. (1999) Structure, function, and regulation of pyruvate carboxylase. Biochem. J. 340, 1–16. Knowles, J. (1989) The mechanism of biotin-dependent enzymes. Annu. Rev. Biochem. 58, 195–221. Matte, A., Tari, L.W., Goldie, H., & Delbaere, L.T.J. (1997) Structure and mechanism of phosphoenolpyruvate carboxykinase. J. Biol. Chem. 272, 8105–8108. Ovadi, J. & Srere, P. (2000) Macromolecular compartmentation and channeling. Int. Rev. Cytol. 192, 255–280. Advanced review of the evidence for channeling and metabolons. Remington, S.J. (1992) Structure and mechanism of citrate synthase. Curr. Top. Cell. Regul. 33, 209–229. A thorough review of this enzyme. Singer, T.P. & Johnson, M.K. (1985) The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification. FEBS Lett. 190, 189–198. A description of the structure and role of the iron-sulfur centers in this enzyme. Weigand, G. & Remington, S.J. (1986) Citrate synthase: struc- ture, control, and mechanism. Annu. Rev. Biophys. Biophys. Chem. 15, 97–117. Wolodko, W.T., Fraser, M.E., James, M.N.G., & Bridger, W.A. (1994) The crystal structure of succinyl-CoA synthetase from Es- cherichia coli at 2.5-? resolution. J. Biol. Chem. 269, 10,883–10,890. Regulation of the Citric Acid Cycle Hansford, R.G. (1980) Control of mitochondrial substrate oxida- tion. Curr. Top. Bioenerget. 10, 217–278. A detailed review of the regulation of the citric acid cycle. Kaplan, N.O. (1985) The role of pyridine nucleotides in regulating cellular metabolism. Curr. Top. Cell. Regul. 26, 371–381. An excellent general discussion of the importance of the [NADH]/[NAD H11001 ] ratio in cellular regulation. Reed, L.J., Damuni, Z., & Merryfield, M.L. (1985) Regulation of mammalian pyruvate and branched-chain H9251-keto acid dehydro- genase complexes by phosphorylation-dephosphorylation. Curr. Top. Cell. Regul. 27, 41–49. Glyoxylate Cycle Eastmond, P.J. & Graham, I.A. (2001) Re-examining the role of the glyoxylate cycle in oilseeds. Trends Plant Sci. 6, 72–77. Intermediate-level review of studies of the glyoxylate cycle in Arabidopsis. Holms, W.H. (1986) The central metabolic pathways of Es- cherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. Curr. Top. Cell. Regul. 28, 69–106. (d) Write a balanced net equation for the catabolism of acetyl-CoA to CO 2 . 2. Recognizing Oxidation and Reduction Reactions One biochemical strategy of many living organisms is the step- wise oxidation of organic compounds to CO 2 and H 2 O and the conservation of a major part of the energy thus produced in the form of ATP. It is important to be able to recognize oxi- dation-reduction processes in metabolism. Reduction of an organic molecule results from the hydrogenation of a double bond (Eqn 1, below) or of a single bond with accompanying cleavage (Eqn 2). Conversely, oxidation results from dehy- drogenation. In biochemical redox reactions, the coenzymes NAD and FAD dehydrogenate/hydrogenate organic molecules in the presence of the proper enzymes. 1. Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, H9251-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. (a) Write a balanced equation for the reaction catalyzed by each enzyme. (b) Name the cofactor(s) required by each enzyme re- action. (c) For each enzyme determine which of the following describes the type of reaction(s) catalyzed: condensation (carbon–carbon bond formation); dehydration (loss of wa- ter); hydration (addition of water); decarboxylation (loss of CO 2 ); oxidation-reduction; substrate-level phosphorylation; isomerization. Problems O Acetaldehyde reduction G CCH 3 A G C O O H11002 CH 3 B O HH H11001 H11001 B O HH H H11001 OOO H C H11001CH 3 H oxidation reduction oxidation Acetate O Ethanol reduction G CCH 3 A C O CH 3 B OO H H H H11001 B H H H O O AA O OH C (2) CH 3 H oxidation reduction oxidation Acetaldehyde H11001 G O H11002 H D B O O H H (1) 8885d_c16_601-630 1/27/04 8:54 AM Page 627 mac76 mac76:385_reb: Chapter 16 The Citric Acid Cycle628 For each of the metabolic transformations in (a) through (h), determine whether oxidation or reduction has occurred. Bal- ance each transformation by inserting HOH and, where nec- essary, H 2 O. substrates in the presence of the appropriate dehydrogenase. In these reactions, NADH H11001 H H11001 serves as the hydrogen source, as described in Problem 2. Whenever the coenzyme is oxidized, a substrate must be simultaneously reduced: For each of the reactions in (a) through (f), determine whether the substrate has been oxidized or reduced or is un- changed in oxidation state (see Problem 2). If a redox change has occurred, balance the reaction with the necessary amount of NAD H11001 , NADH, H H11001 , and H 2 O. The objective is to recognize when a redox coenzyme is necessary in a metabolic reaction. Acetate G O H11002 H H11001 H H11001 H11001 CO 2 O G D B P M J C CH 2 B G O B O C O H11002 O O H11002 B C CH 3 O O G O B C O CH 3 Succinate Pyruvate M D O O O C O H11002 CH 3 CH 2 C M D O H11002 O H11002 O C O D C P C G H G C G O H11002 O Toluene Fumarate O C A A O H C A H H J O H11002 Benzoate CH 2 OO OC (h) OH H CH 2 OC H A Methanol O A A OH OH O Glycerol Dihydroxyacetone CH 2 O B C A OH CH 2 O A OOH CH 2 OC H A OH CH 2 AA A OH OH J G C O H11002 O A A H OH C G O H GlyceraldehydeGlycerate P O B O C C O H O H11001 H H11001 Carbon dioxide Formaldehyde B O O H11002 G HC O Formate OOH Formaldehyde Formate O B CH O O H OH (a) (b) (c) (d) (e) (f) (g) H11001 H H11001 H11001 H H11001 OO O A H Ethanol OH J G O O Acetaldehyde C Glyceraldehyde 3-phosphate C H OH(a) (b) (c) (d) (e) (f ) J G CH 2 CH 3 CH 3 A C O Malate H H11001 HPO 4 CH 2 O A OH C H AA 2H11002 3 OPO C OPO 3 2H11002 1,3-Bisphosphoglycerate O 2H11002 CH 2 OO A C Oxaloacetate COO H11002 J G H H11001 CO 2 OC Acetaldehyde O H11002 J G CH 3 OO CC Pyruvate O H11002O M D CH 3 O Acetone OH H11001 CO 2 O Acetoacetate O H11002 OOC O B B O CH 2 OO C COO H11002 O H11002 OOC O B A H H11001 CO 2 OC Acetate O H11002 J G CH 3 OO CC Pyruvate H11001 H H11001 O M D CH 3 O B O CH 3 OC J G CH 2 OO C O H11002 O B CH 3 OCC 3 O H11002 CH 2 A 2H11002 3 OPO 3. Relationship between Energy Release and the Oxi- dation State of Carbon A eukaryotic cell can use glucose (C 6 H 12 O 6 ) and hexanoic acid (C 6 H 14 O 2 ) as fuels for cellular respiration. On the basis of their structural formulas, which substance releases more energy per gram on complete com- bustion to CO 2 and H 2 O? 4. Nicotinamide Coenzymes as Reversible Redox Car- riers The nicotinamide coenzymes (see Fig. 13-15) can un- dergo reversible oxidation-reduction reactions with specific Substrate H11001 NADH H11001 H H11001 34 product H11001 NAD H11001 Oxidized Reduced Reduced Oxidized 5. Stimulation of Oxygen Consumption by Oxaloac- etate and Malate In the early 1930s, Albert Szent Gy?rgyi reported the interesting observation that the addition of small amounts of oxaloacetate or malate to suspensions of minced pigeon-breast muscle stimulated the oxygen consumption of the preparation. Surprisingly, the amount of oxygen consumed was about seven times more than the amount necessary for complete oxidation (to CO 2 and H 2 O) of the added oxaloac- etate or malate. Why did the addition of oxaloacetate or malate stimulate oxygen consumption? Why was the amount of oxy- gen consumed so much greater than the amount necessary to completely oxidize the added oxaloacetate or malate? 8885d_c16_628 1/30/04 11:48 AM Page 628 mac76 mac76:385_reb: Chapter 16 Problems 629 6. Formation of Oxaloacetate in a Mitochondrion In the last reaction of the citric acid cycle, malate is dehydro- genated to regenerate the oxaloacetate necessary for the en- try of acetyl-CoA into the cycle: L-Malate H11001 NAD H11001 On oxaloacetate H11001 NADH H11001 H H11001 H9004GH11032H11034 H11005 30.0 kJ/mol (a) Calculate the equilibrium constant for this reaction at 25 H11034C. (b) Because H9004GH11032H11034 assumes a standard pH of 7, the equi- librium constant calculated in (a) corresponds to KH11032 eq H11005 The measured concentration of L-malate in rat liver mito- chondria is about 0.20 mM when [NAD H11001 ]/[NADH] is 10. Cal- culate the concentration of oxaloacetate at pH 7 in these mitochondria. (c) To appreciate the magnitude of the mitochondrial oxaloacetate concentration, calculate the number of ox- aloacetate molecules in a single rat liver mitochondrion. As- sume the mitochondrion is a sphere of diameter 2.0 H9262m. 7. Energy Yield from the Citric Acid Cycle The reac- tion catalyzed by succinyl-CoA synthetase produces the high- energy compound GTP. How is the free energy contained in GTP incorporated into the cellular ATP pool? 8. Respiration Studies in Isolated Mitochondria Cel- lular respiration can be studied in isolated mitochondria by measuring oxygen consumption under different conditions. If 0.01 M sodium malonate is added to actively respiring mito- chondria that are using pyruvate as fuel source, respiration soon stops and a metabolic intermediate accumulates. (a) What is the structure of this intermediate? (b) Explain why it accumulates. (c) Explain why oxygen consumption stops. (d) Aside from removal of the malonate, how can this inhibition of respiration be overcome? Explain. 9. Labeling Studies in Isolated Mitochondria The metabolic pathways of organic compounds have often been delineated by using a radioactively labeled substrate and fol- lowing the fate of the label. (a) How can you determine whether glucose added to a suspension of isolated mitochondria is metabolized to CO 2 and H 2 O? (b) Suppose you add a brief pulse of [3- 14 C]pyruvate (la- beled in the methyl position) to the mitochondria. After one turn of the citric acid cycle, what is the location of the 14 C in the oxaloacetate? Explain by tracing the 14 C label through the pathway. How many turns of the cycle are required to re- lease all the [3- 14 C]pyruvate as CO 2 ? 10. [1- 14 C]Glucose Catabolism An actively respiring bacterial culture is briefly incubated with [1- 14 C] glucose, and the glycolytic and citric acid cycle intermediates are isolated. Where is the 14 C in each of the intermediates listed below? Consider only the initial incorporation of 14 C, in the first pass of labeled glucose through the pathways. (a) Fructose 1,6-bisphosphate (b) Glyceraldehyde 3-phosphate [oxaloacetate][NADH] H5007H5007H5007 [L-malate][NAD H11001 ] (c) Phosphoenolpyruvate (d) Acetyl-CoA (e) Citrate (f ) H9251-Ketoglutarate (g) Oxaloacetate 11. Role of the Vitamin Thiamine People with beriberi, a disease caused by thiamine deficiency, have elevated levels of blood pyruvate and H9251-ketoglutarate, especially after consuming a meal rich in glucose. How are these effects related to a deficiency of thiamine? 12. Synthesis of Oxaloacetate by the Citric Acid Cycle Oxaloacetate is formed in the last step of the citric acid cy- cle by the NAD H11001 -dependent oxidation of L-malate. Can a net synthesis of oxaloacetate from acetyl-CoA occur using only the enzymes and cofactors of the citric acid cycle, without depleting the intermediates of the cycle? Explain. How is ox- aloacetate that is lost from the cycle (to biosynthetic reac- tions) replenished? 13. Mode of Action of the Rodenticide Fluoroacetate Fluoroacetate, prepared commercially for rodent control, is also produced by a South African plant. After entering a cell, fluoroacetate is converted to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase: The toxic effect of fluoroacetate was studied in an experiment using intact isolated rat heart. After the heart was perfused with 0.22 mM fluoroacetate, the measured rate of glucose uptake and glycolysis decreased, and glucose 6-phosphate and fructose 6-phosphate accumulated. Examination of the citric acid cycle intermediates revealed that their concentra- tions were below normal, except for citrate, with a concen- tration 10 times higher than normal. (a) Where did the block in the citric acid cycle occur? What caused citrate to accumulate and the other cycle inter- mediates to be depleted? (b) Fluoroacetyl-CoA is enzymatically transformed in the citric acid cycle. What is the structure of the end prod- uct of fluoroacetate metabolism? Why does it block the citric acid cycle? How might the inhibition be overcome? (c) In the heart perfusion experiments, why did glucose uptake and glycolysis decrease? Why did hexose monophos- phates accumulate? (d) Why is fluoroacetate poisoning fatal? 14. Synthesis of L-Malate in Wine Making The tartness of some wines is due to high concentrations of L-malate. Write a sequence of reactions showing how yeast cells synthesize L-malate from glucose under anaerobic conditions in the pres- ence of dissolved CO 2 (HCO 3 H11002 ). Note that the overall reaction for this fermentation cannot involve the consumption of nicotinamide coenzymes or citric acid cycle intermediates. 15. Net Synthesis of H9251-Ketoglutarate H9251-Ketoglutarate plays a central role in the biosynthesis of several amino acids. Write a sequence of enzymatic reactions that could result in F O H11001 H11001H11001 H11001CH 2 COO H11002 CoA-SH ATP CH 2 C S-CoA AMP PP i F 8885d_c16_601-630 1/27/04 8:54 AM Page 629 mac76 mac76:385_reb: Chapter 16 The Citric Acid Cycle630 the net synthesis of H9251-ketoglutarate from pyruvate. Your pro- posed sequence must not involve the net consumption of other citric acid cycle intermediates. Write an equation for the overall reaction and identify the source of each reactant. 16. Regulation of the Pyruvate Dehydrogenase Com- plex In animal tissues, the rate of conversion of pyruvate to acetyl-CoA is regulated by the ratio of active, phosphory- lated to inactive, unphosphorylated PDH complex. Determine what happens to the rate of this reaction when a preparation of rabbit muscle mitochondria containing the PDH complex is treated with (a) pyruvate dehydrogenase kinase, ATP, and NADH; (b) pyruvate dehydrogenase phosphatase and Ca 2H11001 ; (c) malonate. 17. Commercial Synthesis of Citric Acid Citric acid is used as a flavoring agent in soft drinks, fruit juices, and many other foods. Worldwide, the market for citric acid is valued at hundreds of millions of dollars per year. Commercial pro- duction uses the mold Aspergillus niger, which metabolizes sucrose under carefully controlled conditions. (a) The yield of citric acid is strongly dependent on the concentration of FeCl 3 in the culture medium, as indicated in the graph. Why does the yield decrease when the concentra- tion of Fe 3H11001 is above or below the optimal value of 0.5 mg/L? (b) Write the sequence of reactions by which A. niger synthesizes citric acid from sucrose. Write an equation for the overall reaction. (c) Does the commercial process require the culture medium to be aerated—that is, is this a fermentation or an aerobic process? Explain. 18. Regulation of Citrate Synthase In the presence of saturating amounts of oxaloacetate, the activity of citrate syn- thase from pig heart tissue shows a sigmoid dependence on the concentration of acetyl-CoA, as shown in the graph. When succinyl-CoA is added, the curve shifts to the right and the sigmoid dependence is more pronounced. 1 2 3 4 5 90 80 70 60 50 Y ield of citric acid (%) [FeCl 3 ] (mg/L) On the basis of these observations, suggest how succinyl-CoA regulates the activity of citrate synthase. (Hint: See Fig. 6–29.) Why is succinyl-CoA an appropriate signal for regulation of the citric acid cycle? How does the regulation of citrate synthase control the rate of cellular respiration in pig heart tissue? 19. Regulation of Pyruvate Carboxylase The carboxy- lation of pyruvate by pyruvate carboxylase occurs at a very low rate unless acetyl-CoA, a positive allosteric modulator, is present. If you have just eaten a meal rich in fatty acids (tri- acylglycerols) but low in carbohydrates (glucose), how does this regulatory property shut down the oxidation of glucose to CO 2 and H 2 O but increase the oxidation of acetyl-CoA de- rived from fatty acids? 20. Relationship between Respiration and the Citric Acid Cycle Although oxygen does not participate directly in the citric acid cycle, the cycle operates only when O 2 is present. Why? 21. Thermodynamics of Citrate Synthase Reaction in Cells Citrate is formed by the condensation of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase: Oxaloacetate H11001 acetyl-CoA H11001 H 2 O On citrate H11001 CoA H11001 H H11001 In rat heart mitochondria at pH 7.0 and 25 H11034C, the concen- trations of reactants and products are: oxaloacetate, 1 H9262M; acetyl-CoA, 1 H9262M; citrate, 220 H9262M; and CoA, 65 H9262M. The stan- dard free-energy change for the citrate synthase reaction is H1100232.2 kJ/mol. What is the direction of metabolite flow through the citrate synthase reaction in rat heart cells? Explain. 22. Reactions of the Pyruvate Dehydrogenase Complex Two of the steps in the oxidative decarboxylation of pyruvate (steps 4 and 5 in Fig. 16–6) do not involve any of the three carbons of pyruvate yet are essential to the operation of the PDH complex. Explain. No succinyl-CoA Activity (% of V max ) 100 80 60 40 20 20 40 60 80 100 120 [Acetyl-CoA] ( M) Succinyl-CoA added H9262 8885d_c16_630 1/30/04 11:48 AM Page 630 mac76 mac76:385_reb: chapter T he oxidation of long-chain fatty acids to acetyl-CoA is a central energy-yielding pathway in many organ- isms and tissues. In mammalian heart and liver, for example, it provides as much as 80% of the energetic needs under all physiological circumstances. The elec- trons removed from fatty acids during oxidation pass through the respiratory chain, driving ATP synthesis; the acetyl-CoA produced from the fatty acids may be completely oxidized to CO 2 in the citric acid cycle, re- sulting in further energy conservation. In some species and in some tissues, the acetyl-CoA has alternative fates. In liver, acetyl-CoA may be converted to ketone bod- ies—water-soluble fuels exported to the brain and other tissues when glucose is not available. In higher plants, acetyl-CoA serves primarily as a biosynthetic precursor, only secondarily as fuel. Although the biological role of fatty acid oxidation differs from organism to organism, the mechanism is essentially the same. The repetitive four-step process, called H9252 oxidation, by which fatty acids are converted into acetyl-CoA is the main topic of this chapter. In Chapter 10 we described the properties of tria- cylglycerols (also called triglycerides or neutral fats) that make them especially suitable as storage fuels. The long alkyl chains of their constituent fatty acids are es- sentially hydrocarbons, highly reduced structures with an energy of complete oxidation (~38 kJ/g) more than twice that for the same weight of carbohydrate or pro- tein. This advantage is compounded by the extreme insolubility of lipids in water; cellular triacylglycerols aggregate in lipid droplets, which do not raise the osmolarity of the cytosol, and they are unsolvated. (In storage polysaccharides, by contrast, water of solvation can account for two-thirds of the overall weight of the stored molecules.) And because of their relative chem- ical inertness, triacylglycerols can be stored in large quantity in cells without the risk of undesired chemical reactions with other cellular constituents. The properties that make triacylglycerols good stor- age compounds, however, present problems in their role as fuels. Because they are insoluble in water, ingested triacylglycerols must be emulsified before they can be digested by water-soluble enzymes in the intestine, and triacylglycerols absorbed in the intestine or mobilized from storage tissues must be carried in the blood bound to proteins that counteract their insolubility. To over- come the relative stability of the COC bonds in a fatty acid, the carboxyl group at C-1 is activated by attach- ment to coenzyme A, which allows stepwise oxidation of the fatty acyl group at the C-3, or H9252, position—hence the name H9252 oxidation. We begin this chapter with a brief discussion of the sources of fatty acids and the routes by which they travel to the site of their oxidation, with special emphasis on the process in vertebrates. We then describe the chem- ical steps of fatty acid oxidation in mitochondria. The complete oxidation of fatty acids to CO 2 and H 2 O takes place in three stages: the oxidation of long-chain fatty acids to two-carbon fragments, in the form of acetyl-CoA (H9252 oxidation); the oxidation of acetyl-CoA to CO 2 in the citric acid cycle (Chapter 16); and the transfer of FATTY ACID CATABOLISM 17.1 Digestion, Mobilization, and Transport of Fats 632 17.2 Oxidation of Fatty Acids 637 17.3 Ketone Bodies 650 Jack Sprat could eat no fat, His wife could eat no lean, And so between them both you see, They licked the platter clean. —John Clarke, Paroemiologia Anglo-Latina (Proverbs English and Latin), 1639 17 631 electrons from reduced electron carriers to the mitochon- drial respiratory chain (Chapter 19). In this chapter we focus on the first of these stages. We begin our discus- sion of H9252 oxidation with the simple case in which a fully saturated fatty acid with an even number of carbon atoms is degraded to acetyl-CoA. We then look briefly at the extra transformations necessary for the degrada- tion of unsaturated fatty acids and fatty acids with an odd number of carbons. Finally, we discuss variations on the H9252-oxidation theme in specialized organelles— peroxisomes and glyoxysomes—and two less common pathways of fatty acid catabolism, H9275 and H9251 oxidation. The chapter concludes with a description of an alternative fate for the acetyl-CoA formed by H9252 oxidation in verte- brates: the production of ketone bodies in the liver. 17.1 Digestion, Mobilization, and Transport of Fats Cells can obtain fatty acid fuels from three sources: fats consumed in the diet, fats stored in cells as lipid droplets, and fats synthesized in one organ for export to another. Some species use all three sources under various circumstances, others use one or two. Verte- brates, for example, obtain fats in the diet, mobilize fats stored in specialized tissue (adipose tissue, consisting of cells called adipocytes), and, in the liver, convert ex- cess dietary carbohydrates to fats for export to other tissues. On average, 40% or more of the daily energy re- quirement of humans in highly industrialized countries is supplied by dietary triacylglycerols (although most nutritional guidelines recommend no more than 30% of daily caloric intake from fats). Triacylglycerols provide more than half the energy requirements of some organs, particularly the liver, heart, and resting skeletal muscle. Stored triacylglycerols are virtually the sole source of energy in hibernating animals and migrating birds. Pro- tists obtain fats by consuming organisms lower in the food chain, and some also store fats as cytosolic lipid droplets. Vascular plants mobilize fats stored in seeds during germination, but do not otherwise depend on fats for energy. Dietary Fats Are Absorbed in the Small Intestine In vertebrates, before ingested triacylglycerols can be absorbed through the intestinal wall they must be con- verted from insoluble macroscopic fat particles to finely dispersed microscopic micelles. This solubilization is carried out by bile salts, such as taurocholic acid (p. 355), which are synthesized from cholesterol in the liver, stored in the gallbladder, and released into the small intestine after ingestion of a fatty meal. Bile salts are amphipathic compounds that act as biological deter- gents, converting dietary fats into mixed micelles of bile salts and triacylglycerols (Fig. 17–1, step 1 ). Micelle formation enormously increases the fraction of lipid molecules accessible to the action of water-soluble lipases in the intestine, and lipase action converts tria- cylglycerols to monoacylglycerols (monoglycerides) and diacylglycerols (diglycerides), free fatty acids, and glyc- erol (step 2 ). These products of lipase action diffuse into the epithelial cells lining the intestinal surface (the intestinal mucosa) (step 3 ), where they are recon- verted to triacylglycerols and packaged with dietary cholesterol and specific proteins into lipoprotein aggre- gates called chylomicrons (Fig. 17–2; see also Fig. 17–1, step 4 ). Apolipoproteins are lipid-binding proteins in the blood, responsible for the transport of triacylglycerols, phospholipids, cholesterol, and cholesteryl esters be- tween organs. Apolipoproteins (“apo” means “detached” or “separate,” designating the protein in its lipid-free form) combine with lipids to form several classes of lipoprotein particles, spherical aggregates with hy- drophobic lipids at the core and hydrophilic protein side chains and lipid head groups at the surface. Various combinations of lipid and protein produce particles of different densities, ranging from chylomicrons and very- low-density lipoproteins (VLDL) to very-high-density lipoproteins (VHDL), which can be separated by ultra- centrifugation. The structures of these lipoprotein par- ticles and their roles in lipid transport are detailed in Chapter 21. The protein moieties of lipoproteins are recognized by receptors on cell surfaces. In lipid uptake from the intestine, chylomicrons, which contain apolipoprotein C-II (apoC-II), move from the intestinal mucosa into the lymphatic system, and then enter the blood, which car- ries them to muscle and adipose tissue (Fig. 17–1, step 5 ). In the capillaries of these tissues, the extracellular enzyme lipoprotein lipase, activated by apoC-II, hy- drolyzes triacylglycerols to fatty acids and glycerol (step 6 ), which are taken up by cells in the target tissues (step 7 ). In muscle, the fatty acids are oxidized for en- ergy; in adipose tissue, they are reesterified for storage as triacylglycerols (step 8 ). The remnants of chylomicrons, depleted of most of their triacylglycerols but still containing cholesterol and apolipoproteins, travel in the blood to the liver, where they are taken up by endocytosis, mediated by recep- tors for their apolipoproteins. Triacylglycerols that en- ter the liver by this route may be oxidized to provide energy or to provide precursors for the synthesis of ke- tone bodies, as described in Section 17.3. When the diet contains more fatty acids than are needed immediately for fuel or as precursors, the liver converts them to triacylglycerols, which are packaged with specific apolipoproteins into VLDLs. The VLDLs are transported in the blood to adipose tissues, where the triacylglyc- erols are removed and stored in lipid droplets within adipocytes. Chapter 17 Fatty Acid Catabolism632 17.1 Digestion, Mobilization, and Transport of Fats 633 Fatty acids are oxidized as fuel or reesterified for storage. 8 Lipoprotein lipase, activated by apoC-II in the capillary, converts triacylglycerols to fatty acids and glycerol. 6 5 Chylomicrons move through the lymphatic system and bloodstream to tissues. 7 Triacylglycerols are incorporated, with cholesterol and apolipoproteins, into chylomicrons. 4 Fats ingested in diet Gallbladder Bile salts emulsify dietary fats in the small intestine, forming mixed micelles. Small intestine Intestinal lipases degrade triacylglycerols. Fatty acids and other breakdown products are taken up by the intestinal mucosa and converted into triacylglycerols. Intestinal mucosa Capillary Chylomicron Lipoprotein lipase Fatty acids enter cells. ApoC-II Myocyte or adipocyte ATP CO 2 1 2 3 FIGURE 17–1 Processing of dietary lipids in vertebrates. Digestion and absorption of dietary lipids occur in the small intestine, and the fatty acids released from triacylglycerols are packaged and delivered to muscle and adipose tissues. The eight steps are discussed in the text. FIGURE 17–2 Molecular structure of a chylomicron. The surface is a layer of phospholipids, with head groups facing the aqueous phase. Triacylglycerols sequestered in the interior (yellow) make up more than 80% of the mass. Several apolipoproteins that protrude from the sur- face (B-48, C-III, C-II) act as signals in the uptake and metabolism of chylomicron contents. The diameter of chylomicrons ranges from about 100 to 500 nm. Apolipoproteins B-48 C-III C-II Phospholipids Triacylglycerols and cholesteryl esters Cholesterol Hormones Trigger Mobilization of Stored Triacylglycerols Neutral lipids are stored in adipocytes (and in steroid- synthesizing cells of the adrenal cortex, ovary, and testes) in the form of lipid droplets, with a core of sterol esters and triacylglycerols surrounded by a monolayer of phospholipids. The surface of these droplets is coated with perilipins, a family of proteins that restrict access to lipid droplets, preventing untimely lipid mobilization. When hormones signal the need for metabolic energy, triacylglycerols stored in adipose tissue are mobilized (brought out of storage) and transported to tissues (skeletal muscle, heart, and renal cortex) in which fatty acids can be oxidized for energy production. The hor- mones epinephrine and glucagon, secreted in response to low blood glucose levels, activate the enzyme adenylyl cyclase in the adipocyte plasma membrane (Fig. 17–3), which produces the intracellular second messenger cyclic AMP (cAMP; see Fig. 12–13). Cyclic AMP– dependent protein kinase (PKA) phosphorylates perilipin A, and the phosphorylated perilipin causes hormone-sensitive lipase in the cytosol to move to the lipid droplet surface, where it can begin hydrolyz- ing triacylglycerols to free fatty acids and glycerol. PKA also phosphorylates hormone-sensitive lipase, doubling or tripling its activity, but the more than 50-fold increase in fat mobilization triggered by epinephrine is due pri- marily to perilipin phosphorylation. Cells with defective perilipin genes have almost no response to increases in cAMP concentration; their hormone-sensitive lipase does not associate with lipid droplets. As hormone-sensitive lipase hydrolyzes triacylglyc- erol in adipocytes, the fatty acids thus released (free fatty acids, FFA) pass from the adipocyte into the blood, where they bind to the blood protein serum al- bumin. This protein (M r 66,000), which makes up about half of the total serum protein, noncovalently binds as many as 10 fatty acids per protein monomer. Bound to this soluble protein, the otherwise insoluble fatty acids are carried to tissues such as skeletal muscle, heart, and renal cortex. In these target tissues, fatty acids dissoci- ate from albumin and are moved by plasma membrane transporters into cells to serve as fuel. About 95% of the biologically available energy of tri- acylglycerols resides in their three long-chain fatty acids; only 5% is contributed by the glycerol moiety. The glyc- erol released by lipase action is phosphorylated by glyc- erol kinase (Fig. 17–4), and the resulting glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate. The glycolytic enzyme triose phosphate isomerase con- verts this compound to glyceraldehyde 3-phosphate, which is oxidized via glycolysis. Fatty Acids Are Activated and Transported into Mitochondria The enzymes of fatty acid oxidation in animal cells are located in the mitochondrial matrix, as demonstrated in 1948 by Eugene P. Kennedy and Albert Lehninger. The fatty acids with chain lengths of 12 or fewer carbons enter mitochondria without the help of membrane trans- porters. Those with 14 or more carbons, which consti- tute the majority of the FFA obtained in the diet or released from adipose tissue, cannot pass directly through the mitochondrial membranes—they must first undergo the three enzymatic reactions of the carnitine shuttle. The first reaction is catalyzed by a family of isozymes (different isozymes specific for fatty acids hav- ing short, intermediate, or long carbon chains) present Chapter 17 Fatty Acid Catabolism634 Bloodstream P P P P P P Adipocyte Myocyte Lipid droplet Perilipin cAMP PKA ATP Hormone CO 2 Serum albumin ATP Fatty acid transporter Adenylyl cyclase Fatty acids 1 2 6 8 7 5 3 4 b oxidation, citric acid cycle, respiratory chain Triacyl- glycerol P Hormone- sensitive lipase Receptor G 5 FIGURE 17–3 Mobilization of triacylglycerols stored in adipose tis- sue. When low levels of glucose in the blood trigger the release of glucagon, 1 the hormone binds its receptor in the adipocyte mem- brane and thus 2 stimulates adenylyl cyclase, via a G protein, to produce cAMP. This activates PKA, which phosphorylates 3 the hormone-sensitive lipase and 4 perilipin molecules on the surface of the lipid droplet. Phosphorylation of perilipin permits hormone- sensitive lipase access to the surface of the lipid droplet, where 5 it hydrolyzes triacylglycerols to free fatty acids. 6 Fatty acids leave the adipocyte, bind serum albumin in the blood, and are carried in the blood; they are released from the albumin and 7 enter a myocyte via a specific fatty acid transporter. 8 In the myocyte, fatty acids are oxidized to CO 2 , and the energy of oxidation is conserved in ATP, which fuels muscle contraction and other energy requiring metabo- lism in the myocyte. in the outer mitochondrial membrane, the acyl-CoA synthetases, which promote the general reaction Fatty acid H11001 CoA H11001 ATP 8z y8 fatty acyl–CoA H11001 AMP H11001 PP i Thus, acyl-CoA synthetases catalyze the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A to yield a fatty acyl–CoA, coupled to the cleavage of ATP to AMP and PP i . (Recall the description of this reaction in Chapter 13, to illustrate how the free energy released by cleav- age of phosphoanhydride bonds in ATP could be cou- pled to the formation of a high-energy compound; p. XXX.) The reaction occurs in two steps and involves a fatty acyl–adenylate intermediate (Fig. 17–5). Fatty acyl–CoAs, like acetyl-CoA, are high-energy compounds; their hydrolysis to FFA and CoA has a large, negative standard free-energy change (H9004GH11032H11034 ≈ H1100231 kJ/mol). The formation of a fatty acyl–CoA is made more favorable by the hydrolysis of two high-energy bonds in ATP; the pyrophosphate formed in the activation reaction is immediately hydrolyzed by inorganic pyrophosphatase (left side of Fig. 17–5), which pulls the preceding activa- tion reaction in the direction of fatty acyl–CoA formation. The overall reaction is Fatty acid H11001 CoA H11001 ATP On fatty acyl–CoA H11001 AMP H11001 2P i (17–1) H9004GH11032H11034 H11005 H1100234 kJ/mol Fatty acyl–CoA esters formed at the cytosolic side of the outer mitochondrial membrane can be trans- ported into the mitochondrion and oxidized to produce ATP, or they can be used in the cytosol to synthesize 17.1 Digestion, Mobilization, and Transport of Fats 635 CH 2 HO C H P O H11002 CH 2 OH HO C H CH 2 OH O O H11002 O CH 2 C P O H11002 CH 2 OH O O H11002 O O CH 2 C P O H11002 O O H11002 O COHOH H Glycerol ATP ADP NADH H11001 H H11001 NAD H11001 D-Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate L-Glycerol 3-phosphate glycerol kinase Glycolysis triose phosphate isomerase glycerol 3-phosphate dehydrogenase CH 2 OH FIGURE 17–4 Entry of glycerol into the glycolytic pathway. 2P i P H11002 O O O O H11002 P O O O H11002 P O O O H11002 Adenosine R O H11002 O C Fatty acyl–CoA fatty acyl–CoA synthetase Fatty acid P OO Adenosine O H11002 O O R C H11001 H11002 O P O O O H11002 P O O H11002 O H11002 AMP inorganic pyrophosphatase O R C Fatty acyl–adenylate (enzyme-bound) S-CoA CoA-SH Pyrophosphate fatty acyl–CoA synthetase ATP H9004GH11032H11034 H11005 H1100219 kJ/mol H9004GH11032H11034 H11005 H1100215 kJ/mol (for the two-step process) 1 2 MECHANISM FIGURE 17–5 Conversion of a fatty acid to a fatty acyl–CoA. The conversion is catalyzed by fatty acyl–CoA synthetase and inorganic pyrophosphatase. Fatty acid activation by formation of the fatty acyl–CoA derivative occurs in two steps. In step H17033 1, the carboxylate ion displaces the outer two (H9252 and H9253) phosphates of ATP to form a fatty acyl–adenylate, the mixed anhydride of a carboxylic acid and a phosphoric acid. The other product is PP i , an excellent leaving group that is immediately hydrolyzed to two P i , pulling the reaction in the forward direction. In step H17033 2, the thiol group of coenzyme A carries out nucleophilic attack on the enzyme-bound mixed anhydride, displacing AMP and forming the thioester fatty acyl–CoA. The overall reaction is highly exergonic. membrane lipids. Fatty acids destined for mitochondrial oxidation are transiently attached to the hydroxyl group of carnitine to form fatty acyl–carnitine—the second reaction of the shuttle. This transesterification is cat- alyzed by carnitine acyltransferase I (M r 88,000), in the outer membrane. Either the acyl-CoA passes through the outer membrane and is converted to the carnitine ester in the intermembrane space (Fig. 17–6), or the carnitine ester is formed on the cytosolic face of the outer membrane, then moved across the outer mem- brane to the intermembrane space—the current evi- dence does not reveal which. In either case, passage into the intermembrane space (the space between the outer and inner membranes) occurs through large pores (formed by the protein porin) in the outer membrane. The fatty acyl–carnitine ester then enters the matrix by facilitated diffusion through the acyl-carnitine/carni- tine transporter of the inner mitochondrial membrane (Fig. 17–6). In the third and final step of the carnitine shuttle, the fatty acyl group is enzymatically transferred from carnitine to intramitochondrial coenzyme A by carni- tine acyltransferase II. This isozyme, located on the inner face of the inner mitochondrial membrane, re- generates fatty acyl–CoA and releases it, along with free carnitine, into the matrix (Fig. 17–6). Carnitine reen- ters the intermembrane space via the acyl-carnitine/car- nitine transporter. CH 2 CH CH 2 CH 3 N H11001 CH 3 COO H11002 Carnitine CH 3 OH This three-step process for transferring fatty acids into the mitochondrion—esterification to CoA, transes- terification to carnitine followed by transport, and trans- esterification back to CoA—links two separate pools of coenzyme A and of fatty acyl–CoA, one in the cytosol, the other in mitochondria. These pools have different functions. Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids, and some amino acids, whereas cytosolic coen- zyme A is used in the biosynthesis of fatty acids (see Fig. 21–10). Fatty acyl–CoA in the cytosolic pool can be used for membrane lipid synthesis or can be moved into the mitochondrial matrix for oxidation and ATP pro- duction. Conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate. The carnitine-mediated entry process is the rate- limiting step for oxidation of fatty acids in mitochondria and, as discussed later, is a regulation point. Once in- side the mitochondrion, the fatty acyl–CoA is acted upon by a set of enzymes in the matrix. SUMMARY 17.1 Digestion, Mobilization, and Transport of Fats ■ The fatty acids of triacylglycerols furnish a large fraction of the oxidative energy in animals. Dietary triacylglycerols are emulsified in the small intestine by bile salts, hydrolyzed by intestinal lipases, absorbed by intestinal epithelial cells, reconverted into triacylglycerols, then formed into chylomicrons by combination with specific apolipoproteins. Chapter 17 Fatty Acid Catabolism636 Cytosol R C O R C O Matrix Carnitine Transporter Carnitine S-CoA CoA-SH Carnitine acyltransferase II Carnitine Carnitine R C O R C O S-CoA CoA-SH Carnitine acyltransferase I Intermembrane space Inner mitochondrial membrane Outer mitochondrial membrane FIGURE 17–6 Fatty acid entry into mitochondria via the acyl-carnitine/ carnitine transporter. After fatty acyl–carnitine is formed at the outer membrane or in the intermembrane space, it moves into the matrix by facilitated diffusion through the transporter in the inner membrane. In the matrix, the acyl group is transferred to mitochondrial coenzyme A, freeing carnitine to return to the intermembrane space through the same transporter. Acyltransferase I is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis (see Fig. 21–1). This inhibition prevents the simultaneous synthesis and degradation of fatty acids. ■ Chylomicrons deliver triacylglycerols to tissues, where lipoprotein lipase releases free fatty acids for entry into cells. Triacylglycerols stored in adipose tissue are mobilized by a hormone-sensitive triacylglycerol lipase. The released fatty acids bind to serum albumin and are carried in the blood to the heart, skeletal muscle, and other tissues that use fatty acids for fuel. ■ Once inside cells, fatty acids are activated at the outer mitochondrial membrane by conversion to fatty acyl–CoA thioesters. Fatty acyl–CoA to be oxidized enters mitochondria in three steps, via the carnitine shuttle. 17.2 Oxidation of Fatty Acids As noted earlier, mitochondrial oxidation of fatty acids takes place in three stages (Fig. 17–7). In the first stage—H9252 oxidation—fatty acids undergo oxidative re- moval of successive two-carbon units in the form of acetyl-CoA, starting from the carboxyl end of the fatty acyl chain. For example, the 16-carbon palmitic acid (palmitate at pH 7) undergoes seven passes through the oxidative sequence, in each pass losing two carbons as acetyl-CoA. At the end of seven cycles the last two car- bons of palmitate (originally C-15 and C-16) remain as acetyl-CoA. The overall result is the conversion of the 16-carbon chain of palmitate to eight two-carbon acetyl groups of acetyl-CoA molecules. Formation of each acetyl-CoA requires removal of four hydrogen atoms (two pairs of electrons and four H H11001 ) from the fatty acyl moiety by dehydrogenases. In the second stage of fatty acid oxidation, the acetyl groups of acetyl-CoA are oxidized to CO 2 in the citric acid cycle, which also takes place in the mito- chondrial matrix. Acetyl-CoA derived from fatty acids thus enters a final common pathway of oxidation with the acetyl-CoA derived from glucose via glycolysis and pyruvate oxidation (see Fig. 16–1). The first two stages of fatty acid oxidation produce the reduced electron car- riers NADH and FADH 2 , which in the third stage donate electrons to the mitochondrial respiratory chain, through which the electrons pass to oxygen with the concomitant phosphorylation of ADP to ATP (Fig. 17–7). The energy released by fatty acid oxidation is thus conserved as ATP. We now take a closer look at the first stage of fatty acid oxidation, beginning with the simple case of a sat- urated fatty acyl chain with an even number of carbons, then turning to the slightly more complicated cases of unsaturated and odd-number chains. We also consider the regulation of fatty acid oxidation, the H9252-oxidative processes as they occur in organelles other than mito- chondria, and, finally, two less-general modes of fatty acid catabolism, H9251 oxidation and H9275 oxidation. The H9252 Oxidation of Saturated Fatty Acids Has Four Basic Steps Four enzyme-catalyzed reactions make up the first stage of fatty acid oxidation (Fig. 17–8a). First, dehydro- genation of fatty acyl–CoA produces a double bond between the H9251 and H9252 carbon atoms (C-2 and C-3), yield- ing a trans-H9004 2 -enoyl-CoA (the symbol H9004 2 designates the position of the double bond; you may want to re- view fatty acid nomenclature, p. 343.) Note that the new double bond has the trans configuration, whereas the double bonds in naturally occurring unsaturated fatty acids are normally in the cis configuration. We consider the significance of this difference later. This first step is catalyzed by three isozymes of acyl-CoA dehydrogenase, each specific for a range of fatty-acyl chain lengths: very-long-chain acyl-CoA de- hydrogenase (VLCAD), acting on fatty acids of 12 to 18 17.2 Oxidation of Fatty Acids 637 NADH, FADH 2 Respiratory (electron-transfer) chain ATP H 2 O 2H H11001 H11001 1 O 2 Stage 1 Stage 2 Stage 3 16CO 2 Citric acid cycle 8 Acetyl-CoA e H5008 e H5008 H11001 P i 2 CH 2 64e H5008 CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C CH 2 O H5008 O H9252 Oxidation ADP FIGURE 17–7 Stages of fatty acid oxidation. Stage 1: A long-chain fatty acid is oxidized to yield acetyl residues in the form of acetyl- CoA. This process is called H9252 oxidation. Stage 2: The acetyl groups are oxidized to CO 2 via the citric acid cycle. Stage 3: Electrons derived from the oxidations of stages 1 and 2 pass to O 2 via the mitochon- drial respiratory chain, providing the energy for ATP synthesis by oxidative phosphorylation. carbons; medium-chain (MCAD), acting on fatty acids of 4 to 14 carbons; and short-chain (SCAD), acting on fatty acids of 4 to 8 carbons. All three isozymes are flavo- proteins with FAD (see Fig. 13–18) as a prosthetic group. The electrons removed from the fatty acyl–CoA are transferred to FAD, and the reduced form of the de- hydrogenase immediately donates its electrons to an electron carrier of the mitochondrial respiratory chain, the electron-transferring flavoprotein (ETF) (see Fig. 19–8). The oxidation catalyzed by an acyl-CoA de- hydrogenase is analogous to succinate dehydrogenation in the citric acid cycle (p. XXX); in both reactions the enzyme is bound to the inner membrane, a double bond is introduced into a carboxylic acid between the H9251 and H9252 carbons, FAD is the electron acceptor, and electrons from the reaction ultimately enter the respiratory chain and pass to O 2 , with the concomitant synthesis of about 1.5 ATP molecules per electron pair. In the second step of the H9252-oxidation cycle (Fig. 17–8a), water is added to the double bond of the trans-H9004 2 -enoyl-CoA to form the L stereoisomer of H9252-hydroxyacyl-CoA (3-hydroxyacyl-CoA). This re- action, catalyzed by enoyl-CoA hydratase, is for- mally analogous to the fumarase reaction in the citric acid cycle, in which H 2 O adds across an H9251–H9252 double bond (p. XXX). In the third step, L-H9252-hydroxyacyl-CoA is dehydro- genated to form H9252-ketoacyl-CoA, by the action of H9252-hydroxyacyl-CoA dehydrogenase; NAD H11001 is the electron acceptor. This enzyme is absolutely specific for the L stereoisomer of hydroxyacyl-CoA. The NADH formed in the reaction donates its electrons to NADH dehydrogenase, an electron carrier of the respiratory chain, and ATP is formed from ADP as the electrons pass to O 2 . The reaction catalyzed by H9252-hydroxyacyl-CoA de- hydrogenase is closely analogous to the malate dehy- drogenase reaction of the citric acid cycle (p. XXX). The fourth and last step of the H9252-oxidation cycle is catalyzed by acyl-CoA acetyltransferase, more com- monly called thiolase, which promotes reaction of H9252- ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA. The other product is the coenzyme A thioester of the fatty acid, now short- ened by two carbon atoms (Fig. 17–8a). This reaction is called thiolysis, by analogy with the process of hy- drolysis, because the H9252-ketoacyl-CoA is cleaved by re- action with the thiol group of coenzyme A. The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the en- zymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of H9251 4 H9252 4 subunits. Each H9251 subunit contains two activities, the enoyl-CoA hydratase and the H9252-hydroxyacyl-CoA dehydrogenase; the H9252 subunits contain the thiolase ac- tivity. This tight association of three enzymes may allow efficient substrate channeling from one active site to the Chapter 17 Fatty Acid Catabolism638 CH 2 CH 3 O CH 2 (C 14 ) Acyl-CoA (myristoyl-CoA) H9251 S-CoA H9252 acyl-CoA CoA-SH C C CH 2 S-CoA C O Palmitoyl-CoA acyl-CoA FADH 2 FAD H 2 O RCH 2 O trans-H9004 2 - Enoyl-CoA S-CoACCC H CH 2 H enoyl-CoA RCH 2 O L-H9252-Hydroxy- acyl-CoA S-CoA H9252-hydroxyacyl-CoA NADH H11001 H H11001 NAD H11001 CC OH H CH 2 O (C 16 ) R RCH 2 O H9252-Ketoacyl-CoA S-CoACCCH 2 O S-CoA H11001 (b) Acetyl (C 14 ) R C 12 C 10 C 8 C 6 C 14 Acetyl -CoA Acetyl -CoA Acetyl -CoA Acetyl -CoA Acetyl -CoA (a) dehydrogenase hydratase dehydrogenase acetyltransferase (thiolase) C 4 -CoA Acetyl -CoA Acetyl -CoA FIGURE 17–8 The H9252-oxidation pathway. (a) In each pass through this four-step sequence, one acetyl residue (shaded in pink) is removed in the form of acetyl-CoA from the carboxyl end of the fatty acyl chain— in this example palmitate (C 16 ), which enters as palmitoyl-CoA. (b) Six more passes through the pathway yield seven more molecules of acetyl-CoA, the seventh arising from the last two carbon atoms of the 16-carbon chain. Eight molecules of acetyl-CoA are formed in all. next, without diffusion of the intermediates away from the enzyme surface. When TFP has shortened the fatty acyl chain to 12 or fewer carbons, further oxidations are catalyzed by a set of four soluble enzymes in the matrix. As noted earlier, the single bond between methyl- ene (OCH 2 O) groups in fatty acids is relatively stable. The H9252-oxidation sequence is an elegant mechanism for destabilizing and breaking these bonds. The first three reactions of H9252 oxidation create a much less stable COC bond, in which the H9251 carbon (C-2) is bonded to two car- bonyl carbons (the H9252-ketoacyl-CoA intermediate). The ketone function on the H9252 carbon (C-3) makes it a good target for nucleophilic attack by the OSH of coenzyme A, catalyzed by thiolase. The acidity of the H9251 hydrogen and the resonance stabilization of the carbanion gener- ated by the departure of this hydrogen make the termi- nal OCH 2 OCOOS-CoA a good leaving group, facilitating breakage of the H9251–H9252 bond. The Four H9252-Oxidation Steps Are Repeated to Yield Acetyl-CoA and ATP In one pass through the H9252-oxidation sequence, one mol- ecule of acetyl-CoA, two pairs of electrons, and four pro- tons (H H11001 ) are removed from the long-chain fatty acyl–CoA, shortening it by two carbon atoms. The equa- tion for one pass, beginning with the coenzyme A ester of our example, palmitate, is Palmitoyl-CoA H11001 CoA H11001 FAD H11001 NAD H11001 H11001 H 2 O On myristoyl-CoA H11001 acetyl-CoA H11001FADH 2 H11001 NADH H11001 H H11001 (17–2) Following removal of one acetyl-CoA unit from palmitoyl- CoA, the coenzyme A thioester of the shortened fatty acid (now the 14-carbon myristate) remains. The myristoyl-CoA can now go through another set of four H9252-oxidation reactions, exactly analogous to the first, to yield a second molecule of acetyl-CoA and lauroyl-CoA, the coenzyme A thioester of the 12-carbon laurate. Altogether, seven passes through the H9252-oxidation sequence are required to oxidize one molecule of palmitoyl-CoA to eight molecules of acetyl-CoA (Fig. 17–8b). The overall equation is Palmitoyl-CoA H11001 7CoA H11001 7FAD H11001 7NAD H11001 H11001 7H 2 O On 8 acetyl-CoA H11001 7FADH 2 H11001 7NADH H11001 7H H11001 (17–3) Each molecule of FADH 2 formed during oxidation of the fatty acid donates a pair of electrons to ETF of the res- piratory chain, and about 1.5 molecules of ATP are gen- erated during the ensuing transfer of each electron pair to O 2 . Similarly, each molecule of NADH formed deliv- ers a pair of electrons to the mitochondrial NADH de- hydrogenase, and the subsequent transfer of each pair of electrons to O 2 results in formation of about 2.5 mol- ecules of ATP. Thus four molecules of ATP are formed for each two-carbon unit removed in one pass through the sequence. Note that water is also produced in this process. Transfer of electrons from NADH or FADH 2 to O 2 yields one H 2 O per electron pair. Reduction of O 2 by NADH also consumes one H H11001 per NADH molecule: NADH H11001 H H11001 H11001 H5007 1 2 H5007 O 2 On NAD H11001 H11001 H 2 O. In hibernating animals, fatty acid oxidation provides metabolic energy, heat, and water—all essential for survival of an animal that neither eats nor drinks for long periods (Box 17–1). Camels obtain water to supplement the meager supply available in their natural environment by oxidation of fats stored in their hump. The overall equation for the oxidation of palmitoyl- CoA to eight molecules of acetyl-CoA, including the electron transfers and oxidative phosphorylations, is Palmitoyl-CoA H11001 7CoA H11001 7O 2 H11001 28P i H11001 28ADP On 8 acetyl-CoA H11001 28ATP H11001 7H 2 O (17–4) Acetyl-CoA Can Be Further Oxidized in the Citric Acid Cycle The acetyl-CoA produced from the oxidation of fatty acids can be oxidized to CO 2 and H 2 O by the citric acid cycle. The following equation represents the balance sheet for the second stage in the oxidation of palmitoyl- CoA, together with the coupled phosphorylations of the third stage: 8 Acetyl-CoA H11001 16O 2 H11001 80P i H11001 80ADP On 8CoA H11001 80ATP H11001 16CO 2 H11001 16H 2 O (17–5) Combining Equations 17–4 and 17–5, we obtain the overall equation for the complete oxidation of palmitoyl- CoA to carbon dioxide and water: Palmitoyl-CoA H11001 23O 2 H11001 108P i H11001 108ADP On CoA H11001 108ATP H11001 16CO 2 H11001 23H 2 O (17–6) Table 17–1 summarizes the yields of NADH, FADH 2 , and ATP in the successive steps of palmitoyl-CoA oxida- tion. Note that because the activation of palmitate to palmitoyl-CoA breaks both phosphoanhydride bonds in ATP (Fig. 17–5), the energetic cost of activating a fatty acid is equivalent to two ATP, and the net gain per mol- ecule of palmitate is 106 ATP. The standard free-energy change for the oxidation of palmitate to CO 2 and H 2 O is about 9,800 kJ/mol. Under standard conditions, the energy recovered as the phosphate bond energy of ATP is 106 H11003 30.5 kJ/mol H11005 3,230 kJ/mol, about 33% of the theoretical maximum. However, when the free-energy changes are calculated from actual concentrations of re- actants and products under intracellular conditions (see Box 13–1), the free-energy recovery is more than 60%; the energy conservation is remarkably efficient. Oxidation of Unsaturated Fatty Acids Requires Two Additional Reactions The fatty acid oxidation sequence just described is typ- ical when the incoming fatty acid is saturated (that is, has only single bonds in its carbon chain). However, 17.2 Oxidation of Fatty Acids 639 most of the fatty acids in the triacylglycerols and phos- pholipids of animals and plants are unsaturated, having one or more double bonds. These bonds are in the cis configuration and cannot be acted upon by enoyl-CoA hydratase, the enzyme catalyzing the addition of H 2 O to the trans double bond of the H9004 2 -enoyl-CoA generated during H9252 oxidation. Two auxiliary enzymes are needed for H9252 oxidation of the common unsaturated fatty acids: an isomerase and a reductase. We illustrate these aux- iliary reactions with two examples. Chapter 17 Fatty Acid Catabolism640 BOX 17–1 THE WORLD OF BIOCHEMISTRY Fat Bears Carry Out H9252 Oxidation in Their Sleep Many animals depend on fat stores for energy during hibernation, during migratory periods, and in other sit- uations involving radical metabolic adjustments. One of the most pronounced adjustments of fat metabo- lism occurs in hibernating grizzly bears. These animals remain in a continuous state of dormancy for periods as long as seven months. Unlike most hibernating species, the bear maintains a body temperature of be- tween 32 and 35 H11034C, close to the normal (nonhiber- nating) level. Although expending about 25,000 kJ/day (6,000 kcal/day), the bear does not eat, drink, urinate, or defecate for months at a time. Experimental studies have shown that hibernat- ing grizzly bears use body fat as their sole fuel. Fat oxidation yields sufficient energy for maintenance of body temperature, active synthesis of amino acids and proteins, and other energy-requiring activities, such as membrane transport. Fat oxidation also re- leases large amounts of water, as described in the text, which replenishes water lost in breathing. The glyc- erol released by degradation of triacylglycerols is con- verted into blood glucose by gluconeogenesis. Urea formed during breakdown of amino acids is reab- sorbed in the kidneys and recycled, the amino groups reused to make new amino acids for maintaining body proteins. Bears store an enormous amount of body fat in preparation for their long sleep. An adult grizzly con- sumes about 38,000 kJ/day during the late spring and summer, but as winter approaches it feeds 20 hours a day, consuming up to 84,000 kJ daily. This change in feeding is a response to a seasonal change in hormone secretion. Large amounts of triacylglycerols are formed from the huge intake of carbohydrates during the fattening-up period. Other hibernating species, in- cluding the tiny dormouse, also accumulate large amounts of body fat. A grizzly bear prepares its hibernation nest, near the McNeil River in Canada. Number of NADH Number of ATP Enzyme catalyzing the oxidation step or FADH 2 formed ultimately formed* Acyl-CoA dehydrogenase 7 FADH 2 10.5 H9252-Hydroxyacyl-CoA dehydrogenase 7 NADH 17.5 Isocitrate dehydrogenase 8 NADH 20 H9251-Ketoglutarate dehydrogenase 8 NADH 20 Succinyl-CoA synthetase 8 ? Succinate dehydrogenase 8 FADH 2 12 Malate dehydrogenase 8 NADH 20 Total 108 TABLE 17–1 Yield of ATP during Oxidation of One Molecule of Palmitoyl-CoA to CO 2 and H 2 O * These calculations assume that mitochondrial oxidative phosphorylation produces 1.5 ATP per FADH 2 oxidized and 2.5 ATP per NADH oxidized. ? GTP produced directly in this step yields ATP in the reaction catalyzed by nucleoside diphosphate kinase (p. XXX). Oleate is an abundant 18-carbon monounsaturated fatty acid with a cis double bond between C-9 and C-10 (denoted H9004 9 ). In the first step of oxidation, oleate is con- verted to oleoyl-CoA and, like the saturated fatty acids, enters the mitochondrial matrix via the carnitine shut- tle (Fig. 17–6). Oleoyl-CoA then undergoes three passes through the fatty acid oxidation cycle to yield three mol- ecules of acetyl-CoA and the coenzyme A ester of a H9004 3 , 12-carbon unsaturated fatty acid, cis-H9004 3 -dodecenoyl- CoA (Fig. 17–9). This product cannot serve as a sub- strate for enoyl-CoA hydratase, which acts only on trans double bonds. The auxiliary enzyme H9004 3 ,H9004 2 -enoyl-CoA isomerase isomerizes the cis-H9004 3 -enoyl-CoA to the trans-H9004 2 -enoyl-CoA, which is converted by enoyl-CoA hydratase into the corresponding L-H9252-hydroxyacyl-CoA (trans-H9004 2 -dodecenoyl-CoA). This intermediate is now acted upon by the remaining enzymes of H9252 oxidation to yield acetyl-CoA and the coenzyme A ester of a 10- carbon saturated fatty acid, decanoyl-CoA. The latter undergoes four more passes through the pathway to yield five more molecules of acetyl-CoA. Altogether, nine acetyl-CoAs are produced from one molecule of the 18-carbon oleate. The other auxiliary enzyme (a reductase) is re- quired for oxidation of polyunsaturated fatty acids—for example, the 18-carbon linoleate, which has a cis-H9004 9 ,cis- H9004 12 configuration (Fig. 17–10). Linoleoyl-CoA under- goes three passes through the H9252-oxidation sequence to yield three molecules of acetyl-CoA and the coenzyme A ester of a 12-carbon unsaturated fatty acid with a cis- H9004 3 ,cis-H90046 configuration. This intermediate cannot be used by the enzymes of the H9252-oxidation pathway; its double bonds are in the wrong position and have the wrong configuration (cis, not trans). However, the com- bined action of enoyl-CoA isomerase and 2,4-dienoyl- CoA reductase, as shown in Figure 17–10, allows reen- try of this intermediate into the H9252-oxidation pathway 17.2 Oxidation of Fatty Acids 641 18 1 9 S-CoA O C oxidationb (five cycles) S-CoA H C H O H9004 3 , H9004 2 -enoyl-CoA isomerase S-CoA H C O H H9252 oxidation (three cycles) 3 Acetyl-CoA 6 Acetyl-CoA Oleoyl-CoA trans-H9004 2 - Dodecenoyl-CoA cis-H9004 3 - Dodecenoyl-CoA 12 12 CoA oxidation (one cycle, and first oxidation of second cycle) Acetyl- NADPH H11001 H H11001 NADP H11001 enoyl-CoA isomerase 3 18 C 9 6 12 2(a) CoA oxidation (four cycles) 3 Acetyl- 1 4 2 5 3 2,4-dienoyl-CoA reductase H9004 3 , H9004 2 -enoyl-CoA isomerase oxidation (three cycles) 5 Acetyl-CoA Linoleoyl-CoA trans-H9004 2 cis-H9004 9 , cis-H9004 12 cis-H9004 3 , cis-H9004 6 trans-H9004 2 , , cis-H9004 6 trans-H9004 2 cis-H9004 4 trans-H9004 3 S-CoA C O S-CoA C O 2(a)5 5 3(b) 3(b) 4 6 4 C S-CoA O S-CoA O 1 S-CoA C O 4 1 2 5 3 S-CoA C O 4 1 2 12 12 10 10 10 b b b FIGURE 17–9 Oxidation of a monounsaturated fatty acid. Oleic acid, as oleoyl-CoA (H9004 9 ), is the example used here. Oxidation requires an additional enzyme, enoyl-CoA isomerase, to reposition the double bond, converting the cis isomer to a trans isomer, a normal interme- diate in H9252 oxidation. FIGURE 17–10 Oxidation of a polyunsaturated fatty acid. The example here is linoleic acid, as linoleoyl-CoA (H9004 9,12 ). Oxidation re- quires a second auxiliary enzyme in addition to enoyl-CoA isomerase: NADPH-dependent 2,4-dienoyl-CoA reductase. The combined action of these two enzymes converts a trans-H9004 2 ,cis-H9004 4 -dienoyl-CoA inter- mediate to the trans-H9004 2 -enoyl-CoA substrate necessary for H9252 oxidation. and its degradation to six acetyl-CoAs. The overall re- sult is conversion of linoleate to nine molecules of acetyl-CoA. Complete Oxidation of Odd-Number Fatty Acids Requires Three Extra Reactions Although most naturally occurring lipids contain fatty acids with an even number of carbon atoms, fatty acids with an odd number of carbons are common in the lipids of many plants and some marine organisms. Cattle and other ruminant animals form large amounts of the three- carbon propionate (CH 3 OCH 2 OCOO H11002 ) during fer- mentation of carbohydrates in the rumen. The propi- onate is absorbed into the blood and oxidized by the liver and other tissues. And small quantities of propi- onate are added as a mold inhibitor to some breads and cereals, thus entering the human diet. Long-chain odd-number fatty acids are oxidized in the same pathway as the even-number acids, beginning at the carboxyl end of the chain. However, the substrate for the last pass through the H9252-oxidation sequence is a fatty acyl–CoA with a five-carbon fatty acid. When this is oxidized and cleaved, the products are acetyl-CoA and propionyl-CoA. The acetyl-CoA can be oxidized in the citric acid cycle, of course, but propionyl-CoA enters a different pathway involving three enzymes. Propionyl-CoA is first carboxylated to form the D stereoisomer of methylmalonyl-CoA (Fig. 17–11) by propionyl-CoA carboxylase, which contains the co- factor biotin. In this enzymatic reaction, as in the pyru- vate carboxylase reaction (see Fig. 16–16), CO 2 (or its hydrated ion, HCO 3 H11002 ) is activated by attachment to bi- otin before its transfer to the substrate, in this case the propionate moiety. Formation of the carboxybiotin in- termediate requires energy, which is provided by the cleavage of ATP to ADP and P i . The D-methylmalonyl- CoA thus formed is enzymatically epimerized to its L stereoisomer by methylmalonyl-CoA epimerase (Fig. 17–11). The L-methylmalonyl-CoA then undergoes an intramolecular rearrangement to form succinyl-CoA, which can enter the citric acid cycle. This rearrange- ment is catalyzed by methylmalonyl-CoA mutase, which requires as its coenzyme 5H11541-deoxyadenosyl- cobalamin, or coenzyme B 12 , which is derived from vitamin B 12 (cobalamin). Box 17–2 describes the role of coenzyme B 12 in this remarkable exchange reaction. Fatty Acid Oxidation Is Tightly Regulated Oxidation of fatty acids consumes a precious fuel, and it is regulated so as to occur only when the need for en- ergy requires it. In the liver, fatty acyl–CoA formed in the cytosol has two major pathways open to it: (1) H9252 ox- idation by enzymes in mitochondria or (2) conversion into triacylglycerols and phospholipids by enzymes in the cytosol. The pathway taken depends on the rate of transfer of long-chain fatty acyl–CoA into mitochondria. The three-step process (carnitine shuttle) by which fatty acyl groups are carried from cytosolic fatty acyl–CoA into the mitochondrial matrix (Fig. 17–6) is rate-limiting for fatty acid oxidation and is an important point of regulation. Once fatty acyl groups have entered the mitochondrion, they are committed to oxidation to acetyl-CoA. Malonyl-CoA, the first intermediate in the cytoso- lic biosynthesis of long-chain fatty acids from acetyl-CoA (see Fig. 21–1), increases in concentration whenever the animal is well supplied with carbohydrate; excess glucose that cannot be oxidized or stored as glycogen is converted in the cytosol into fatty acids for storage as triacylglycerol. The inhibition of carnitine acyltrans- ferase I by malonyl-CoA ensures that the oxidation of Chapter 17 Fatty Acid Catabolism642 H C O HCO 3 H11002 Propionyl-CoA ATP ADP H11001 P i D-Methylmalonyl-CoA L-Methylmalonyl-CoA Succinyl-CoA H11002 O O H11002 propionyl-CoA biotin HC C C H H O H C CoA-S O HC C HH H H C O HC C HH H C OO H11002 HC C HH C O carboxylase C O methylmalonyl-CoA epimerase CoA-S CoA-S CoA-S methyl- malonyl-CoA mutase coenzyme B 12 FIGURE 17–11 Oxidation of propionyl-CoA produced by H9252 oxida- tion of odd-number fatty acids. The sequence involves the carboxy- lation of propionyl-CoA to D-methylmalonyl-CoA and conversion of the latter to succinyl-CoA. This conversion requires epimerization of D- to L-methylmalonyl-CoA, followed by a remarkable reaction in which substituents on adjacent carbon atoms exchange positions (see Box 17–2). fatty acids is inhibited whenever the liver is amply sup- plied with glucose as fuel and is actively making tria- cylglycerols from excess glucose. Two of the enzymes of H9252 oxidation are also regu- lated by metabolites that signal energy sufficiency. When the [NADH]/[NAD H11001 ] ratio is high, H9252-hydroxyacyl- CoA dehydrogenase is inhibited; in addition, high con- centrations of acetyl-CoA inhibit thiolase (Fig. 17–12). Genetic Defects in Fatty Acyl–CoA Dehydrogenases Cause Serious Disease Stored triacylglycerols are typically the chief source of energy for muscle contraction, and an inability to oxidize fatty acids from triacylglycerols has serious consequences for health. The most common ge- netic defect in fatty acid catabolism in U.S. and north- ern European populations is due to a mutation in the gene encoding the medium-chain acyl-CoA dehy- drogenase (MCAD). Among northern Europeans, the H11002 OOC CH 2 S-CoAC O Malonyl-CoA frequency of carriers (individuals with this recessive mutation on one of the two homologous chromosomes) is about 1 in 40, and about 1 individual in 10,000 has the disease—that is, has two copies of the mutant MCAD allele and is unable to oxidize fatty acids of 6 to 12 car- bons. The disease is characterized by recurring episodes of a syndrome that includes fat accumulation in the liver, high blood levels of octanoic acid, low blood glucose (hypoglycemia), sleepiness, vomiting, and coma. The pattern of organic acids in the urine helps in the diag- nosis of this disease: the urine commonly contains high levels of 6-carbon to 10-carbon dicarboxylic acids (pro- duced by H9275 oxidation) and low levels of urinary ketone bodies (we discuss H9275 oxidation below and ketone bod- ies in Section 17.3). Although individuals may have no symptoms between episodes, the episodes are very se- rious; mortality from this disease is 25% to 60% in early childhood. If the genetic defect is detected shortly after birth, the infant can be started on a low-fat, high- carbohydrate diet. With early detection and careful man- agement of the diet—including avoiding long intervals between meals, to prevent the body from turning to its fat reserves for energy—the prognosis for these indi- viduals is good. 17.2 Oxidation of Fatty Acids 643 Fatty acid synthesis Fatty acid b oxidation b oxidation 1 2 6 7 4 8 5 3 Dietary carbohydrate High blood glucose Insulin Inactive Glucagon PKA ACC ACC Low blood glucose Fatty acyl–CoA Carnitine Fatty acyl–CoA CoASH FADH NADH Acetyl-CoA Fatty acyl– carnitine Fatty acyl– carnitine Mitochondrion Acetyl–CoA Malonyl-CoAGlucose Fatty acids carnitine acyl- transferase I multistep glycolysis, pyruvate dehydrogenase complex P phosphatase P i FIGURE 17–12 Coordinated regulation of fatty acid synthesis and breakdown. When the diet provides a ready source of carbohydrate as fuel, H9252 oxidation of fatty acids is unnecessary and is therefore down- regulated. Two enzymes are key to the coordination of fatty acid metabolism: acetyl-CoA carboxylase (ACC), the first enzyme in the synthesis of fatty acids (see Fig. 21–1 ), and carnitine acyl transferase I, which limits the transport of fatty acids into the mitochondrial matrix for H9252 oxidation (see Fig. 17–6). Ingestion of a high-carbohydrate meal raises the blood glucose level and thus 1 triggers the release of in- sulin. 2 Insulin-dependent protein phosphatase dephosphorylates ACC, activating it. 3 ACC catalyzes the formation of malonyl-CoA (the first intermediate of fatty acid synthesis), and 4 malonyl-CoA in- hibits carnitine acyltransferase I, thereby preventing fatty acid entry into the mitochondrial matrix. When blood glucose levels drop between meals, 5 glucagon re- lease activates cAMP-dependent protein kinase (PKA), which 6 phos- phorylates and inactivates ACC. The concentration of malonyl-CoA falls, the inhibition of fatty acid entry into mitochondria is relieved, and 7 fatty acids enter the mitochondrial matrix and 8 become the major fuel. Because glucagon also triggers the mobilization of fatty acids in adipose tissue, a supply of fatty acids begins arriving in the blood. 644 BOX 17–2 WORKING IN BIOCHEMISTRY Coenzyme B 12 : A Radical Solution to a Perplexing Problem In the methylmalonyl-CoA mutase reaction (see Fig. 17–11), the group OCOOS-CoA at C-2 of the original propionate exchanges position with a hydrogen atom at C-3 of the original propionate (Fig. 1a). Coenzyme B 12 is the cofactor for this reaction, as it is for almost all enzymes that catalyze reactions of this general type (Fig. 1b). These coenzyme B 12 –dependent processes are among the very few enzymatic reactions in biol- ogy in which there is an exchange of an alkyl or sub- stituted alkyl group (X) with a hydrogen atom on an adjacent carbon, with no mixing of the transferred hydrogen atom with the hydrogen of the solvent, H 2 O. How can the hydrogen atom move between two carbons without mixing with the enormous excess of hydrogen atoms in the solvent? Coenzyme B 12 is the cofactor form of vitamin B 12 , which is unique among all the vitamins in that it contains not only a complex organic molecule but an essential trace element, cobalt. The com- plex corrin ring system of vitamin B 12 (colored blue in Fig. 2), to which cobalt (as Co 3H11001 ) is coordinated, is chemically re- lated to the porphyrin ring system of heme and heme proteins (see Fig. 5–1). A fifth coordination position of cobalt is filled by dimethylbenzimidazole ribonucleotide (shaded yellow), bound covalently by its 3H11032-phosphate group to a side chain of the corrin ring, through aminoisopropanol. The formation of this complex cofactor oc- curs in one of only two known reactions in which triphosphate is cleaved from ATP (Fig. 3); the other reaction is the forma- tion of S-adenosylmethionine from ATP and methionine (see Fig. 18–18). Vitamin B 12 as usually isolated is called cyanocobalamin, because it contains a cyano group (picked up during purification) attached to cobalt in the sixth coordination position. In 5H11541-deoxyadenosylcobalamin, the cofactor for methylmalonyl-CoA mu- tase, the cyano group is replaced by the 5H11541-deoxyadenosyl group (red in Fig. 2), covalently bound through C-5H11032 to the cobalt. The three-dimensional structure of the co- factor was determined by Dorothy Crowfoot Hodgkin in 1956, using x-ray crystallo- graphy. The key to understanding how coen- zyme B 12 catalyzes hydrogen exchange lies in the properties of the covalent bond be- tween cobalt and C-5H11032 of the deoxyadeno- FIGURE 1 N CH 3 N N N HO 3H11032 1H11032 2H11032 5H11032 4H11032 Co 3H11001 N N N N H H H H H CH 3 CH 3 CH 3 CH 2 CH 2 CH 3 CH 2 CH 2 CH 3 CH 3 CH 2 C NH 2 C O O CH 2 NH CH 2 HC CH 3 O H OH HH H CH 3 C O NH 2 CH 2 H 2 N CH 2 CH 2 CH 2 C O CH 2 O H OH H H CH 3 CH 2 O PO H11002 H N N O O CH 3 OH Corrin ring system Amino- isopropanol 5H11032-Deoxy- adenosine Dimethyl- benzimidazole ribonucleotide NH 2 C O CH 2 NH 2 C O NH 2 C O NH 2 H O H CC O C O H11002 H S-CoA Succinyl-CoA H H O C (b) C O H11002 H S-CoA L-Methylmalonyl-CoA methylmalonyl-CoA mutase coenzyme B 12 coenzyme B 12 O (a) HCC H CC H C XHX CC syl group (Fig. 2). This is a relatively weak bond; its bond dissociation energy is about 110 kJ/mol, com- pared with 348 kJ/mol for a typical COC bond or 414 kJ/mol for a COH bond. Merely illuminating the com- pound with visible light is enough to break this CoOC bond. (This extreme photolability probably accounts for the absence of vitamin B 12 in plants.) Dissociation produces a 5H11032-deoxyadenosyl radical and the Co 2H11001 FIGURE 2 645 form of the vitamin. The chemical function of 5H11032-de- oxyadenosylcobalamin is to generate free radicals in this way, thus initiating a series of transformations such as that illustrated in Figure 4— a postulated mechanism for the reaction catalyzed by methylmalonyl-CoA mutase and a number of other coen- zyme B 12 –dependent trans- formations. 1 The enzyme first breaks the CoOC bond in the cofactor, leaving the coenzyme in its Co 2H11001 form and producing the 5H11032-deoxyadenosyl free radical. 2 This radical now abstracts a hydrogen atom from the sub- strate, converting the substrate to a radical and pro- ducing 5H11032-deoxyadenosine. 3 Rearrangement of the substrate radical yields another radical, in which the migrating group X (OCOOS-CoA for methylmalonyl- CoA mutase) has moved to the adjacent carbon to form a radical that has the carbon skeleton of the eventual product (a four-carbon straight chain). The hydrogen atom initially abstracted from the substrate is now part of the OCH 3 group of 5H11032-deoxyadenosine. 4 One of the hydrogens from this same OCH 3 group (it can be the same one originally abstracted) is now returned to the productlike radical, generating the product and regenerating the deoxyadenosyl free radical. 5 The bond re-forms between cobalt and the OCH 2 group of the deoxyadenosyl radical, destroying the free radical and regenerating the cofactor in its Co 3H11001 form, ready to undergo another catalytic cycle. In this postulated mechanism, the migrating hydrogen atom never exists as a free species and is thus never free to exchange with the hydrogen of surrounding water molecules. Vitamin B 12 deficiency results in serious disease. This vitamin is not made by plants or animals and can be synthesized only by a few species of mi- croorganisms. It is required by healthy people in only minute amounts, about 3 H9262g/day. The severe disease pernicious anemia results from failure to absorb vi- tamin B 12 efficiently from the intestine, where it is synthesized by intestinal bacteria or obtained from di- gestion of meat. Individuals with this disease do not produce sufficient amounts of intrinsic factor, a gly- coprotein essential to vitamin B 12 absorption. The pathology in pernicious anemia includes reduced pro- duction of erythrocytes, reduced levels of hemoglobin, and severe, progressive impairment of the central nerv- ous system. Administration of large doses of vitamin B 12 alleviates these symptoms in at least some cases. ■ FIGURE 3 N N N N P NH 2 O H HO Coenzyme B 12 H H OH CH 2 Co H11002 OOP O O H11002 OP O O H11002 O H11002 H O ATP POOP O O H11002 OP O O H11002 O H11002 O O H11002 O H11002 N N N N 3H11032 1H11032 2H11032 5H11032 4H11032 NH 2 O H HO H H OH CH 2 H Cobalamin Co NN N CH 2 H C H NN N H C H NN N H C H HC C X NN N H C H HC C X 5 1 CC XH Product 5H11032-Deoxyadenosyl free radical Substrate radical Productlike radical 2 C H C X Substrate 3 4 5H11032-Deoxy- adenosyl free radical Coenzyme B 12 Co 2H11001 N Co 3H11001 N Co 2H11001 N Co 2H11001 N NN N Co 2H11001 N Deoxyadenosine Deoxyadenosine Deoxyadenosine Deoxyadenosine Deoxyadenosine radical rearrangement Dorothy Crowfoot Hodgkin, 1910–1994 MECHANISM FIGURE 4 More than 20 other human genetic defects in fatty acid transport or oxidation have been documented, most much less common than the defect in MCAD. One of the most severe disorders results from loss of the long-chain H9252-hydroxyacyl-CoA dehydrogenase activity of the tri- functional protein, TFP. Other disorders include defects in the H9251 or H9252 subunits that affect all three activities of TFP and cause serious heart disease and abnormal skeletal muscle. ■ Peroxisomes Also Carry Out H9252 Oxidation The mitochondrial matrix is the major site of fatty acid oxidation in animal cells, but in certain cells other com- partments also contain enzymes capable of oxidizing fatty acids to acetyl-CoA, by a pathway similar to, but not identical with, that in mitochondria. In plant cells, the major site of H9252 oxidation is not mitochondria but peroxisomes. In peroxisomes, membrane-enclosed organelles of animal and plant cells, the intermediates for H9252 oxidation of fatty acids are coenzyme A derivatives, and the process consists of four steps, as in mitochondrial H9252 ox- idation (Fig. 17–13): (1) dehydrogenation, (2) addition of water to the resulting double bond, (3) oxidation of the H9252-hydroxyacyl-CoA to a ketone, and (4) thiolytic cleavage by coenzyme A. (The identical reactions also occur in glyoxysomes, as discussed below.) One difference between the peroxisomal and mito- chondrial pathways is in the chemistry of the first step. In peroxisomes, the flavoprotein acyl-CoA oxidase that introduces the double bond passes electrons directly to O 2 , producing H 2 O 2 (Fig. 17–13). This strong and po- tentially damaging oxidant is immediately cleaved to H 2 O and O 2 by catalase. Recall that in mitochondria, the electrons removed in the first oxidation step pass through the respiratory chain to O 2 to produce H 2 O, and this process is accompanied by ATP synthesis. In per- oxisomes, the energy released in the first oxidative step of fatty acid breakdown is not conserved as ATP, but is dissipated as heat. A second important difference between mito- chondrial and peroxisomal H9252 oxidation in mam- mals is in the specificity for fatty acyl–CoAs; the peroxisomal system is much more active on very-long- chain fatty acids such as hexacosanoic acid (26:0) and on branched-chain fatty acids such as phytanic acid and pristanic acid (see Fig. 17–17). These less-common fatty acids are obtained in the diet from dairy products, the fat of ruminant animals, meat, and fish. Their catabo- lism in the peroxisome involves several auxiliary en- zymes unique to this organelle. The inability to oxidize these compounds is responsible for several serious hu- man diseases. Individuals with Zellweger syndrome are unable to make peroxisomes and therefore lack all the metabolism unique to that organelle. In X-linked adrenoleukodystrophy (XALD), peroxisomes fail to oxidize very-long-chain fatty acids, apparently for lack of a functional transporter for these fatty acids in the peroxisomal membrane. Both defects lead to accumu- lation in the blood of very-long-chain fatty acids, espe- cially 26:0. XALD affects young boys before the age of 10 years, causing loss of vision, behavioral disturbances, and death within a few years. ■ In mammals, high concentrations of fats in the diet result in increased synthesis of the enzymes of peroxi- somal H9252 oxidation in the liver. Liver peroxisomes do not contain the enzymes of the citric acid cycle and cannot catalyze the oxidation of acetyl-CoA to CO 2 . Instead, Chapter 17 Fatty Acid Catabolism646 C O R S-CoA CoASH CoASH H 2 OH 2 O Mitochondrion Peroxisome/glyoxysome FAD FADH 2 H 2 O 2 O 2 NAD + NADH O 2 H 2 O Respiratory chain FAD ATP FADH 2 O 2 H 2 O Respiratory chain NAD + NADH H 2 O H11001 1 2 O 2 RCH 2 CH 2 C S-CoA O C O CR H C C O CH 2 CR H OH C O CH 2 C O R C O CH 3 H11001 NADH exported for reoxidation H S-CoA S-CoA S-CoA S-CoA ATP Citric acid cycle Acetyl-CoA exported FIGURE 17–13 Comparison of H9252 oxidation in mitochondria and in peroxisomes and glyoxysomes. The peroxisomal/glyoxysomal system differs from the mitochondrial system in two respects: (1) in the first oxidative step electrons pass directly to O 2 , generating H 2 O 2 , and (2) the NADH formed in the second oxidative step cannot be reoxi- dized in the peroxisome or glyoxysome, so reducing equivalents are exported to the cytosol, eventually entering mitochondria. The acetyl- CoA produced by peroxisomes and glyoxysomes is also exported; the acetate from glyoxysomes (organelles found only in germinating seeds) serves as a biosynthetic precursor (see Fig. 17–14). Acetyl-CoA pro- duced in mitochondria is further oxidized in the citric acid cycle. long-chain or branched fatty acids are catabolized to shorter-chain products, such as hexanoyl-CoA, which are exported to mitochondria and completely oxidized. Plant Peroxisomes and Glyoxysomes Use Acetyl-CoA from H9252 Oxidation as a Biosynthetic Precursor In plants, fatty acid oxidation does not occur primarily in mitochondria (as noted earlier) but in the peroxi- somes of leaf tissue and in the glyoxysomes of germi- nating seeds. Plant peroxisomes and glyoxysomes are similar in structure and function; glyoxysomes, which occur only in germinating seeds, may be considered spe- cialized peroxisomes. The biological role of H9252 oxidation in these organelles is to use stored lipids primarily to provide but biosynthetic precursors, not energy. During seed germination, stored triacylglycerols are converted into glucose, sucrose, and a wide variety of essential metabolites (Fig. 17–14). Fatty acids released from the triacylglycerols are first activated to their coen- zyme A derivatives and oxidized in glyoxysomes by the same four-step process that takes place in peroxisomes (Fig. 17–13). The acetyl-CoA produced is converted via the glyoxylate cycle (see Fig. 16–20) to four-carbon precursors for gluconeogenesis (see Fig. 14–18). Gly- oxysomes, like peroxisomes, contain high concentra- tions of catalase, which converts the H 2 O 2 produced by H9252 oxidation to H 2 O and O 2 . The H9252-Oxidation Enzymes of Different Organelles Have Diverged during Evolution Although the H9252-oxidation reactions in mitochondria are essentially the same as those in peroxisomes and gly- oxysomes, the enzymes (isozymes) differ significantly between the two types of organelles. The differences apparently reflect an evolutionary divergence that oc- curred very early, with the separation of gram-positive and gram-negative bacteria (see Fig. 1–6). In mitochondria, the four H9252-oxidation enzymes that act on short-chain fatty acyl–CoAs are separate, soluble proteins (as noted earlier), similar in structure to the analogous enzymes of gram-positive bacteria (Fig. 17–15a). The gram-negative bacteria have four activities in three soluable subunits (Fig. 17–15b), and the eukary- otic enzyme system that acts on long-chain fatty acids— the trifunctional protein, TFP—has three enzyme activ- ities in two subunits that are membrane-associated (Fig. 17–15c). The H9252-oxidation enzymes of plant peroxisomes and glyoxysomes, however, form a complex of proteins, one of which contains four enzymatic activities in a sin- gle polypeptide chain (Fig. 17–15d). The first enzyme, acyl-CoA oxidase, is a single polypeptide chain; the mul- tifunctional protein (MFP) contains the second and third enzyme activities (enoyl-CoA hydratase and hydroxyacyl-CoA dehydrogenase) as well as two auxil- iary activities needed for the oxidation of unsaturated fatty acids (D-3-hydroxyacyl-CoA epimerase and H9004 3 ,H9004 2 - enoyl-CoA isomerase); the fourth enzyme, thiolase, is a separate, soluble polypeptide. It is interesting that the enzymes that catalyze es- sentially the reversal of H9252 oxidation in the synthesis of fatty acids are also organized differently in prokaryotes and eukaryotes; in bacteria, the seven enzymes needed for fatty acid synthesis are separate polypeptides, but in mammals, all seven activities are part of a single, huge polypeptide chain (see Fig. 21–7). One advantage to the cell in having several enzymes of the same pathway en- coded in a single polypeptide chain is that this solves the problem of regulating the synthesis of enzymes that must interact functionally; regulation of the expression of one gene ensures production of the same number of active sites for all enzymes in the path. When each en- zyme activity is on a separate polypeptide, some mech- anism is required to coordinate the synthesis of all the gene products. The disadvantage of having several ac- tivities on the same polypeptide is that the longer the polypeptide chain, the greater is the probability of a mis- take in its synthesis: a single incorrect amino acid in the chain may make all the enzyme activities in that chain useless. Comparison of the gene structures for these pro- teins in many species may shed light on the reasons for the selection of one or the other strategy in evolution. The H9275 Oxidation of Fatty Acids Occurs in the Endoplasmic Reticulum Although mitochondrial H9252 oxidation, in which enzymes act at the carboxyl end of a fatty acid, is by far the most 17.2 Oxidation of Fatty Acids 647 FIGURE 17–14 Triacylglycerols as glucose source in seeds. H9252 Oxi- dation is one stage in a pathway that converts stored triacylglycerols to glucose in germinating seeds. For more detail, see Figure 16–22. Seed triacylglycerols Fatty acids Acetyl-CoA Oxaloacetate Glucose Sucrose, polysaccharides Amino acids Energy Nucleotides Metabolic intermediates lipases H9252 oxidation glyoxylate cycle gluconeogenesis important catabolic fate for fatty acids in animal cells, there is another pathway in some species, including ver- tebrates, that involves oxidation of the H9275 (omega) car- bon—the carbon most distant from the carboxyl group. The enzymes unique to H9275 oxidation are located (in vertebrates) in the endoplasmic reticulum of liver and kidney, and the preferred substrates are fatty acids of 10 or 12 carbon atoms. In mammals H9275 oxidation is nor- mally a minor pathway for fatty acid degradation, but when H9252 oxidation is defective (because of mutation or a carnitine deficiency, for example) it becomes more important. The first step introduces a hydroxyl group onto the H9275 carbon (Fig. 17–16). The oxygen for this group comes from molecular oxygen (O 2 ) in a complex reaction that involves cytochrome P450 and the electron donor NADPH. Reactions of this type are catalyzed by mixed- function oxidases, described in Box 21–1. Two more enzymes now act on the H9275 carbon: alcohol dehydro- genase oxidizes the hydroxyl group to an aldehyde, and aldehyde dehydrogenase oxidizes the aldehyde group to a carboxylic acid, producing a fatty acid with a car- boxyl group at each end. At this point, either end can be attached to coenzyme A, and the molecule can en- Chapter 17 Fatty Acid Catabolism648 Product Product Enz 1 Enz 2 Intermediate Intermediate Intermediate Substrate (a) Gram-positive bacteria and mitochondrial short-chain-specific system (d) Peroxisomal and glyoxysomal system of plants (b) Gram-negative bacteria (c) Mitochondrial very-long- chain-specific system Product Enz 4 Enz 2 Product Inner membrane Enz 3 Enz 1 Enz 1 Enz 1 Matrix Substrate Enz 2 Enz 6 Enz 5 Enz 3 Enz 4 Enz 4 Enz 4 Enz 3 Enz 3 Enz 2 Substrate Substrate MFP FIGURE 17–15 The enzymes of H9252 oxidation. Shown here are the dif- ferent subunit structures of the enzymes of H9252 oxidation in gram-positive and gram-negative bacteria, mitochondria, and plant peroxisomes and glyoxysomes. Enz 1 is acyl-CoA dehydrogenase; Enz 2 , enoyl-CoA hy- dratase; Enz 3 , L-H9252-hydroxyacyl-CoA dehydrogenase; Enz 4 , thiolase; Enz 5 , D-3-hydroxyacyl-CoA epimerase, and Enz 6 , H9004 3 ,H9004 2 -enoyl-CoA iso- merase. (a) The four enzymes of H9252 oxidation in gram-positive bacteria are separate, soluble entities, as are those of the short-chain-specific system of mitochondria. (b) In gram-negative bacteria, the four enzyme activities reside in three polypeptides; enzymes 2 and 3 are parts of a single polypeptide chain. (c) The very-long-chain-specific system of mitochondria is also composed of three polypeptides, one of which includes the activities of enzymes 2 and 3; in this case, the system is bound to the inner mitochondrial membrane. (d) In the peroxisomal and glyoxysomal H9252-oxidation systems of plants, enzymes 1 and 4 are separate polypeptides, but enzymes 2 and 3, as well as two auxiliary enzymes, are part of a single polypeptide chain, the multifunctional pro- tein, MFP. ter the mitochondrion and undergo H9252 oxidation by the normal route. In each pass through the H9252-oxidation pathway, the “double-ended” fatty acid yields dicar- boxylic acids such as succinic acid, which can enter the citric acid cycle, and adipic acid (Fig. 17–16). Phytanic Acid Undergoes H9251 Oxidation in Peroxisomes The presence of a methyl group on the H9252 carbon of a fatty acid makes H9252 oxidation impossible, and these branched fatty acids are catabolized in peroxi- somes of animal cells by H9251 oxidation. In the oxidation of phytanic acid, for example (Fig. 17–17), phytanoyl- CoA is hydroxylated on its H9251 carbon, in a reaction that involves molecular oxygen; decarboxylated to form an aldehyde one carbon shorter; and then oxidized to the 17.2 Oxidation of Fatty Acids 649 O H11002 C(CH 2 ) 10 O CH 3 O H11002 C(CH 2 ) 10 O CH 2 HO NADPH, O 2 mixed-function oxidase NADP H11001 NAD H11001 alcohol dehydrogenase NADH O H11002 CC (CH 2 ) 10 OO H O H11002H11002 O CC (CH 2 ) 10 OO O H11002H11002 O CC (CH 2 ) 2 OO O H11002H11002 O CC (CH 2 ) 4 OO NAD H11001 aldehyde dehydrogenase H9252 oxidation NADH Adipate (adipic acid)Succinate H9275 FIGURE 17–16 The H9275 oxidation of fatty acids in the endoplasmic reticulum. This alternative to H9252 oxidation begins with oxidation of the carbon most distant from the H9251 carbon—the H9275 (omega) carbon. The substrate is usually a medium-chain fatty acid; shown here is lauric acid (laurate). This pathway is generally not the major route for ox- idative catabolism of fatty acids. COOH H9252 phytanoil-CoA synthetase AMP, PP i ATP, CoA-SH Phytanic acid Phytanoyl-CoA phytanoyl-CoA hydroxylase CO S-CoA CO 2 H9251-Ketoglutarate, Ascorbate , Succinate Fe 2H11001 H9251-Hydroxyphytanoyl- CoA CO S-CoA OH H9251-hydroxyphytanoyl- CoA lyase Formic acidFormyl-CoA 2 CO C O H Pristanal aldehyde dehydrogenase NAD(P)H NAD(P) H11001 COOH Pristanic acid H9252 oxidation 4,8,12-Trimethyltri- decanoyl-CoA C O S-CoA Propionyl-CoA H11001 C O S-CoA CH CH 23 FIGURE 17–17 The H9251 oxidation of a branched-chain fatty acid (phy- tanic acid) in peroxisomes. Phytanic acid has a methyl-substituted H9252 carbon and therefore cannot undergo H9252 oxidation. The combined action of the enzymes shown here removes the carboxyl carbon of phytanic acid, to produce pristanic acid, in which the H9252 carbon is unsubstituted, allowing oxidation. Notice that H9252 oxidation of pristanic acid releases propionyl-CoA, not acetyl-CoA. This is further catabo- lized as in Figure 17–11. (The details of the reaction that produces pristanal remain controversial.) corresponding carboxylic acid, which now has no sub- stituent on the H9252 carbon and can be oxidized further by H9252 oxidation. Refsum’s disease, resulting from a genetic defect in phytanoyl-CoA hydroxylase, leads to very high blood levels of phytanic acid and severe neurological problems including blindness and deafness. ■ SUMMARY 17.2 Oxidation of Fatty Acids ■ In the first stage of H9252 oxidation, four reactions remove each acetyl-CoA unit from the carboxyl end of a saturated fatty acyl–CoA: (1) dehydrogenation of the H9251 and H9252 carbons (C-2 and C-3) by FAD-linked acyl-CoA dehydrogenases, (2) hydration of the resulting trans-H9004 2 double bond by enoyl-CoA hydratase, (3) dehydrogenation of the resulting L-H9252-hydroxyacyl-CoA by NAD-linked H9252- hydroxyacyl-CoA dehydrogenase, and (4) CoA-requiring cleavage of the resulting H9252-ketoacyl-CoA by thiolase, to form acetyl-CoA and a fatty acyl–CoA shortened by two carbons. The shortened fatty acyl–CoA then reenters the sequence. ■ In the second stage of fatty acid oxidation, the acetyl-CoA is oxidized to CO 2 in the citric acid cycle. A large fraction of the theoretical yield of free energy from fatty acid oxidation is recovered as ATP by oxidative phosphorylation, the final stage of the oxidative pathway. ■ Malonyl-CoA, an early intermediate of fatty acid synthesis, inhibits carnitine acyltransferase I, preventing fatty acid entry into mitochondria. This blocks fatty acid breakdown while synthesis is occurring. ■ Genetic defects in the medium-chain acyl-CoA dehydrogenase result in serious human disease, as do mutations in other components of the H9252-oxidation system. ■ Oxidation of unsaturated fatty acids requires two additional enzymes: enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. Odd-number fatty acids are oxidized by the H9252-oxidation pathway to yield acetyl-CoA and a molecule of propionyl-CoA. This is carboxylated to methylmalonyl-CoA, which is isomerized to succinyl-CoA in a reaction catalyzed by methylmalonyl-CoA mutase, an enzyme requiring coenzyme B 12 . ■ Peroxisomes of plants and animals, and glyoxysomes of plants, carry out H9252 oxidation in four steps similar to those of the mitochondrial pathway in animals. The first oxidation step, however, transfers electrons directly to O 2 , generating H 2 O 2 . Peroxisomes of animal tissues specialize in the oxidation of very-long-chain fatty acids and branched fatty acids. In glyoxysomes, in germinating seeds, H9252 oxidation is one step in the conversion of stored lipids into a variety of intermediates and products. ■ The reactions of H9275 oxidation, occurring in the endoplasmic reticulum, produce dicarboxylic fatty acyl intermediates, which can undergo H9252 oxidation at either end to yield short dicarboxylic acids such as succinate. 17.3 Ketone Bodies In humans and most other mammals, acetyl-CoA formed in the liver during oxidation of fatty acids can either en- ter the citric acid cycle (stage 2 of Fig. 17–7) or un- dergo conversion to the “ketone bodies,” acetone, ace- toacetate, and D-H9252-hydroxybutyrate, for export to other tissues. (The term “bodies” is a historical artifact; the term is occasionally applied to insoluble particles, but these compounds are quite soluble in blood and urine.) Acetone, produced in smaller quantities than the other ketone bodies, is exhaled. Acetoacetate and D-H9252-hydroxybutyrate are transported by the blood to tis- sues other than the liver (extrahepatic tissues), where they are converted to acetyl-CoA and oxidized in the citric acid cycle, providing much of the energy required by tissues such as skeletal and heart muscle and the renal cortex. The brain, which preferentially uses glu- cose as fuel, can adapt to the use of acetoacetate or D-H9252-hydroxybutyrate under starvation conditions, when glucose is unavailable. The production and export of ke- tone bodies from the liver to extrahepatic tissues allow continued oxidation of fatty acids in the liver when acetyl-CoA is not being oxidized in the citric acid cycle. Ketone Bodies, Formed in the Liver, Are Exported to Other Organs as Fuel The first step in the formation of acetoacetate, occurring in the liver (Fig. 17–18), is the enzymatic condensation of two molecules of acetyl-CoA, catalyzed by thiolase; A O G OH CH 3 C H O H5008 OC A CH 2 O O Acetone B O G CH 3 C O H5008 OC O O O D-H9252-Hydroxybutyrate B CH 3 OC O CH 2 O CH 3 Acetoacetate J J Chapter 17 Fatty Acid Catabolism650 this is simply the reversal of the last step of H9252 oxidation. The acetoacetyl-CoA then condenses with acetyl-CoA to form H9252-hydroxy-H9252-methylglutaryl-CoA (HMG-CoA), which is cleaved to free acetoacetate and acetyl-CoA. The acetoacetate is reversibly reduced by D-H9252-hydroxy- butyrate dehydrogenase, a mitochondrial enzyme, to D-H9252-hydroxybutyrate. This enzyme is specific for the D stereoisomer; it does not act on L-H9252-hydroxyacyl-CoAs and is not to be confused with L-H9252-hydroxyacyl-CoA dehydrogenase of the H9252-oxidation pathway. In healthy people, acetone is formed in very small amounts from acetoacetate, which is easily de- carboxylated, either spontaneously or by the action of acetoacetate decarboxylase (Fig. 17–18). Because individuals with untreated diabetes produce large quan- tities of acetoacetate, their blood contains significant amounts of acetone, which is toxic. Acetone is volatile and imparts a characteristic odor to the breath, which is sometimes useful in diagnosing diabetes. ■ In extrahepatic tissues, D-H9252-hydroxybutyrate is ox- idized to acetoacetate by D-H9252-hydroxybutyrate dehy- drogenase (Fig. 17–19). The acetoacetate is activated to its coenzyme A ester by transfer of CoA from suc- cinyl-CoA, an intermediate of the citric acid cycle (see Fig. 16–7), in a reaction catalyzed by H9252-ketoacyl-CoA transferase. The acetoacetyl-CoA is then cleaved by thiolase to yield two acetyl-CoAs, which enter the citric acid cycle. Thus the ketone bodies are used as fuels. The production and export of ketone bodies by the liver allow continued oxidation of fatty acids with only minimal oxidation of acetyl-CoA. When intermediates of the citric acid cycle are being siphoned off for glucose 17.3 Ketone Bodies 651 OCH 2 O D- -Hydroxybutyrate acetoacetate Acetoacetate D M O C NAD H11001 OCCH 3 S-CoA O 2 Acetyl-CoA G J OCCH 3 S-CoA H11001 G J O C CH 3 S-CoA O Acetoacetyl-CoA G J O C B O O CH 2 OO C CH 3 HMG-CoA CoA-SH Acetyl-CoA H11001H 2 O O C CH 2 S-CoA O A GD M J O C OH CH 3 O H11002 O O CH A H11002 O B O Acetone thiolase CoA-SH HMG-CoA lyase Acetyl-CoA OCH 2 O OH CH 2 OC A CH 3 O CH 3 OCOCH 3 O D M C H11002 O O NADH H11001 H H11001 H9252 H9252 H9252 H9252-Hydroxy- -methylglutaryl-CoA (HMG-CoA) D- -hydroxybutyrate dehydrogenase CO 2 synthase decarboxylase B FIGURE 17–18 Formation of ketone bodies from acetyl-CoA. Healthy, well-nourished individuals produce ketone bodies at a rela- tively low rate. When acetyl-CoA accumulates (as in starvation or un- treated diabetes, for example), thiolase catalyzes the condensation of two acetyl-CoA molecules to acetoacetyl-CoA, the parent compound of the three ketone bodies. The reactions of ketone body formation occur in the matrix of liver mitochondria. The six-carbon compound H9252-hydroxy-H9252-methylglutaryl-CoA (HMG-CoA) is also an intermediate of sterol biosynthesis, but the enzyme that forms HMG-CoA in that pathway is cytosolic. HMG-CoA lyase is present only in the mito- chondrial matrix. FIGURE 17–19 D-H9252-Hydroxybutyrate as a fuel. D-H9252-Hydroxybutyrate, synthesized in the liver, passes into the blood and thus to other tis- sues, where it is converted in three steps to acetyl-CoA. It is first ox- idized to acetoacetate, which is activated with coenzyme A donated from succinyl-CoA, then split by thiolase. The acetyl-CoA thus formed is used for energy production. O CH 3 C H9252-ketoacyl-CoA transferase Succinate D-H9252-Hydroxybutyrate OH CH 2 C H O H11002 CH 3 C O Succinyl-CoA CoA-SH thiolase Acetoacetate NADH H11001 H H11001 NAD H11001 D-H9252-hydroxybutyrate dehydrogenase O CH 2 C O H11002 CH 3 C O H11001 CH 3 C S-CoA O 2 Acetyl-CoA CH 3 C S-CoA O CH 2 C S-CoA O Acetoacetyl-CoA synthesis by gluconeogenesis, for example, oxidation of cycle intermediates slows—and so does acetyl-CoA oxi- dation. Moreover, the liver contains only a limited amount of coenzyme A, and when most of it is tied up in acetyl- CoA, H9252 oxidation slows for want of the free coenzyme. The production and export of ketone bodies free coen- zyme A, allowing continued fatty acid oxidation. Ketone Bodies Are Overproduced in Diabetes and during Starvation Starvation and untreated diabetes mellitus lead to overproduction of ketone bodies, with several associated medical problems. During starvation, gluco- neogenesis depletes citric acid cycle intermediates, di- verting acetyl-CoA to ketone body production (Fig. 17–20). In untreated diabetes, when the insulin level is insufficient, extrahepatic tissues cannot take up glucose efficiently from the blood, either for fuel or for conver- sion to fat. Under these conditions, levels of malonyl- CoA (the starting material for fatty acid synthesis) fall, inhibition of carnitine acyltransferase I is relieved, and fatty acids enter mitochondria to be degraded to acetyl- CoA—which cannot pass through the citric acid cycle because cycle intermediates have been drawn off for use as substrates in gluconeogenesis. The resulting accu- mulation of acetyl-CoA accelerates the formation of ke- tone bodies beyond the capacity of extrahepatic tissues to oxidize them. The increased blood levels of acetoac- etate and D-H9252-hydroxybutyrate lower the blood pH, causing the condition known as acidosis. Extreme acidosis can lead to coma and in some cases death. Ketone bodies in the blood and urine of untreated diabetics can reach extraordinary levels—a blood con- centration of 90 mg/100 mL (compared with a normal level of H110213 mg/100 mL) and urinary excretion of 5,000 mg/24 hr (compared with a normal rate of H11349125 mg/ 24 hr). This condition is called ketosis. Individuals on very low-calorie diets, using the fats stored in adipose tissue as their major energy source, also have increased levels of ketone bodies in their blood and urine. These levels must be monitored to avoid the dangers of acidosis and ketosis (ketoacidosis). ■ SUMMARY 17.3 Ketone Bodies ■ The ketone bodies—acetone, acetoacetate, and D-H9252-hydroxybutyrate—are formed in the liver. The latter two compounds serve as fuel molecules in extrahepatic tissues, through oxidation to acetyl-CoA and entry into the citric acid cycle. ■ Overproduction of ketone bodies in uncontrolled diabetes or severely reduced calorie intake can lead to acidosis or ketosis. Chapter 17 Fatty Acid Catabolism652 FIGURE 17–20 Ketone body formation and export from the liver. Conditions that promote gluconeogenesis (untreated diabetes, severely reduced food intake) slow the citric acid cycle (by drawing off ox- aloacetate) and enhance the conversion of acetyl-CoA to acetoacetate. The released coenzyme A allows continued H9252 oxidation of fatty acids. citric acid cycle Acetyl-CoA H9252 H9252 ketone body formation H9252 CoA Fatty acids Glucose exported as fuel for brain and other tissues Glucose gluconeogenesis Hepatocyte Lipid droplets oxidation Oxaloacetate Acetoacetate, D- -hydroxybutyrate, acetone Acetoacetate and D- -hydroxybutyrate exported as energy source for heart, skeletal muscle, kidney, and brain Key Terms H9252 oxidation XXX chylomicron XXX apolipoprotein XXX lipoprotein XXX perilipins XXX hormone-sensitive lipase XXX free fatty acids XXX serum albumin XXX carnitine shuttle XXX carnitine acyltransferase I XXX acyl-carnitine/carnitine transporter XXX carnitine acyltransferase II XXX trifunctional protein (TFP) XXX methylmalonyl-CoA mutase XXX coenzyme B 12 XXX pernicious anemia XXX intrinsic factor XXX malonyl-CoA XXX medium-chain acyl-CoA dehydrogenase (MCAD) XXX multifunctional protein (MFP) XXX H9275 oxidation XXX mixed-function oxidases XXX H9251 oxidation XXX acidosis XXX ketosis XXX Terms in bold are defined in the glossary. Chapter 17 Problems 653 Further Reading General Boyer, P.D. (1983) The Enzymes, 3rd edn, Vol. 16: Lipid Enzymology, Academic Press, Inc., San Diego, CA. Ferry, G. (1998) Dorothy Hodgkin: A Life, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Fascinating biography of an amazing woman. Gurr, M.I., Harwood, J.L., & Frayn; K.N. (2002) Lipid Biochem- istry: An Introduction, 5th edn, Blackwell Science, Oxford, UK. Langin, D., Holm, C., & Lafontan, M. (1996) Adipocyte hormone-sensitive lipase: a major regulator of lipid metabolism. Proc. Nutr. Soc. 55, 93–109. Ramsay, T.G. (1996) Fat cells. Endocrinol. Metab. Clin. N. Am. 25, 847–870. A review of all aspects of fat storage and mobilization in adipocytes. Scheffler, I.E. (1999) Mitochondria, Wiley-Liss, New York. An excellent book on mitochondrial structure and function. Wang, C.S., Hartsuck, J., & McConathy, W.J. (1992) Structure and functional properties of lipoprotein lipase. Biochim. Biophys. Acta 1123, 1–17. Advanced-level discussion of the enzyme that releases fatty acids from lipoproteins in the capillaries of muscle and adipose tissue. Mitochondrial H9252 Oxidation Bannerjee, R. (1997) The yin-yang of cobalamin biochemistry. Chem. Biol. 4, 175–186. A review of the biochemistry of coenzyme B 12 reactions, includ- ing the methylmalonyl-CoA mutase reaction. Eaton, S., Bartlett, K., & Pourfarzam, M. (1996) Mammalian mitochondrial H9252-oxidation. Biochem. J. 320, 345–357. A review of the enzymology of H9252 oxidation, inherited defects in this pathway, and regulation of the process in mitochondria. Eaton, S., Bursby, T., Middleton, B., Pourfarzam, M., Mills, K., Johnson, A.W., & Bartlett, K. (2000) The mitochondrial trifunctional protein: centre of a H9252-oxidation metabolon? Biochem. Soc. Trans. 28, 177–182. Short, intermediate-level review. Harwood, J.L. (1988) Fatty acid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 101–138. Jeukendrup, A.E., Saris, W.H., & Wagenmakers, A.J. (1998) Fat metabolism during exercise: a review. Part III: effects of nutri- tional interventions. Int. J. Sports Med. 19, 371–379. This paper is one of a series that reviews the factors that influ- ence fat mobilization and utilization during exercise. Kerner, J. & Hoppel, C. (1998) Genetic disorders of carnitine metabolism and their nutritional management. Annu. Rev. Nutr. 18, 179–206. Kerner, J. & Hoppel, C. (2000) Fatty acid import into mitochon- dria. Biochim. Biophys. Acta 1486, 1–17. Kunau, W.H., Dommes, V., & Schulz, H. (1995) H9252-Oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. Prog. Lipid Res. 34, 267–342. A good historical account and a useful comparison of H9252 oxida- tion in different systems. Rinaldo, P., Matern, D., & Bennett, M.J. (2002) Fatty acid oxidation disorders. Annu. Rev. Physiol. 64, 477–502. Advanced review of metabolic defects in fat oxidation, including MCAD mutations. Sherratt, H.S. (1994) Introduction: the regulation of fatty acid oxidation in cells. Biochem. Soc. Trans. 22, 421–422. Introduction to reviews (in this journal issue) of various aspects of fatty acid oxidation and its regulation. Thorpe, C. & Kim, J.J. (1995) Structure and mechanism of action of the acyl-CoA dehydrogenases. FASEB J. 9, 718–725. Short, clear description of the three-dimensional structure and catalytic mechanism of these enzymes. Peroxisomal H9252 Oxidation Graham, I.A. & Eastmond, P.J. (2002) Pathways of straight and branched chain fatty acid catabolism in higher plants. Prog. Lipid Res. 41, 156–181. Hashimoto, T. (1996) Peroxisomal H9252-oxidation: enzymology and molecular biology. Ann. N. Y. Acad. Sci. 804, 86–98. Mannaerts, G.P. & van Veldhoven, P.P. (1996) Functions and organization of peroxisomal H9252-oxidation. Ann. N. Y. Acad. Sci. 804, 99–115. Wanders, R.J.A., van Grunsven, E.G., & Jansen, G.A. (2000) Lipid metabolism in peroxisomes: enzymology, functions and dys- functions of the fatty acid H9251- and H9252-oxidation systems in humans. Biochem. Soc. Trans. 28, 141–148. Ketone Bodies Foster, D.W. & McGarry, J.D. (1983) The metabolic derange- ments and treatment of diabetic ketoacidosis. N. Engl. J. Med. 309, 159–169. McGarry, J.D. & Foster, D.W. (1980) Regulation of hepatic fatty acid oxidation and ketone body production. Annu. Rev. Biochem. 49, 395–420. Robinson, A.M. & Williamson, D.H. (1980) Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol. Rev. 60, 143–187. 1. Energy in Triacylglycerols On a per-carbon basis, where does the largest amount of biologically available en- ergy in triacylglycerols reside: in the fatty acid portions or the glycerol portion? Indicate how knowledge of the chemi- cal structure of triacylglycerols provides the answer. 2. Fuel Reserves in Adipose Tissue Triacylglycerols, with their hydrocarbon-like fatty acids, have the highest en- ergy content of the major nutrients. (a) If 15% of the body mass of a 70.0 kg adult consists of triacylglycerols, what is the total available fuel reserve, in Problems Chapter 17 Fatty Acid Catabolism654 both kilojoules and kilocalories, in the form of triacylglyc- erols? Recall that 1.00 kcal H11005 4.18 kJ. (b) If the basal energy requirement is approximately 8,400 kJ/day (2,000 kcal/day), how long could this person sur- vive if the oxidation of fatty acids stored as triacylglycerols were the only source of energy? (c) What would be the weight loss in pounds per day under such starvation conditions (1 lb H11005 0.454 kg)? 3. Common Reaction Steps in the Fatty Acid Oxida- tion Cycle and Citric Acid Cycle Cells often use the same enzyme reaction pattern for analogous metabolic con- versions. For example, the steps in the oxidation of pyruvate to acetyl-CoA and of H9251-ketoglutarate to succinyl-CoA, al- though catalyzed by different enzymes, are very similar. The first stage of fatty acid oxidation follows a reaction sequence closely resembling a sequence in the citric acid cycle. Use equations to show the analogous reaction sequences in the two pathways. 4. Chemistry of the Acyl-CoA Synthetase Reaction Fatty acids are converted to their coenzyme A esters in a re- versible reaction catalyzed by acyl-CoA synthetase: (a) The enzyme-bound intermediate in this reaction has been identified as the mixed anhydride of the fatty acid and adenosine monophosphate (AMP), acyl-AMP: Write two equations corresponding to the two steps of the reaction catalyzed by acyl-CoA synthetase. (b) The acyl-CoA synthetase reaction is readily re- versible, with an equilibrium constant near 1. How can this reaction be made to favor formation of fatty acyl–CoA? 5. Oxidation of Tritiated Palmitate Palmitate uniformly labeled with tritium ( 3 H) to a specific activity of 2.48 H11003 10 8 counts per minute (cpm) per micromole of palmitate is added to a mitochondrial preparation that oxidizes it to acetyl-CoA. The acetyl-CoA is isolated and hydrolyzed to acetate. The specific activity of the isolated acetate is 1.00 H11003 10 7 cpm/H9262mol. Is this result consistent with the H9252-oxidation path- way? Explain. What is the final fate of the removed tritium? 6. Compartmentation in H9252 Oxidation Free palmitate is activated to its coenzyme A derivative (palmitoyl-CoA) in the cytosol before it can be oxidized in the mitochondrion. If palmitate and [ 14 C]coenzyme A are added to a liver ho- mogenate, palmitoyl-CoA isolated from the cytosolic fraction is radioactive, but that isolated from the mitochondrial frac- tion is not. Explain. 7. Comparative Biochemistry: Energy-Generating Pathways in Birds One indication of the relative impor- tance of various ATP-producing pathways is the V max of cer- tain enzymes of these pathways. The values of V max of sev- eral enzymes from the pectoral muscles (chest muscles used for flying) of pigeon and pheasant are listed below. (a) Discuss the relative importance of glycogen metab- olism and fat metabolism in generating ATP in the pectoral muscles of these birds. (b) Compare oxygen consumption in the two birds. (c) Judging from the data in the table, which bird is the long-distance flyer? Justify your answer. (d) Why were these particular enzymes selected for comparison? Would the activities of triose phosphate iso- merase and malate dehydrogenase be equally good bases for comparison? Explain. 8. Effect of Carnitine Deficiency An individual developed a condition characterized by progressive muscular weakness and aching muscle cramps. The symp- toms were aggravated by fasting, exercise, and a high-fat diet. The homogenate of a skeletal muscle specimen from the patient oxidized added oleate more slowly than did control homogenates, consisting of muscle specimens from healthy individuals. When carnitine was added to the patient’s mus- cle homogenate, the rate of oleate oxidation equaled that in the control homogenates. The patient was diagnosed as hav- ing a carnitine deficiency. (a) Why did added carnitine increase the rate of oleate oxidation in the patient’s muscle homogenate? (b) Why were the patient’s symptoms aggravated by fasting, exercise, and a high-fat diet? (c) Suggest two possible reasons for the deficiency of muscle carnitine in this individual. 9. Fatty Acids as a Source of Water Contrary to legend, camels do not store water in their humps, which actually con- sist of large fat deposits. How can these fat deposits serve as a source of water? Calculate the amount of water (in liters) that a camel can produce from 1.0 kg of fat. Assume for sim- plicity that the fat consists entirely of tripalmitoylglycerol. 10. Petroleum as a Microbial Food Source Some mi- croorganisms of the genera Nocardia and Pseudomonas can grow in an environment where hydrocarbons are the only food source. These bacteria oxidize straight-chain aliphatic hy- drocarbons, such as octane, to their corresponding carboxylic acids: CH 3 (CH 2 ) 6 CH 3 H11001 NAD H11001 H11001 O 2 CH 3 (CH 2 ) 6 COOH H11001 NADH H11001 H H11001 How could these bacteria be used to clean up oil spills? What z y V max (H9262mol substrate/min/g tissue) Enzyme Pigeon Pheasant Hexokinase 3.0 2.3 Glycogen phosphorylase 18.0 120.0 Phosphofructokinase-1 24.0 143.0 Citrate synthase 100.0 15.0 Triacylglycerol lipase 0.07 0.01 ORPC O OO O Adenine HH HH OH CH 2 OH O H11002 R COO H11002 H11001 ATP H11001 CoA CoA H11001 AMP H11001 PP i R C O Chapter 17 Problems 655 would be some of the limiting factors to the efficiency of this process? 11. Metabolism of a Straight-Chain Phenylated Fatty Acid A crystalline metabolite was isolated from the urine of a rabbit that had been fed a straight-chain fatty acid con- taining a terminal phenyl group: A 302 mg sample of the metabolite in aqueous solution was completely neutralized by 22.2 mL of 0.100 M NaOH. (a) What is the probable molecular weight and structure of the metabolite? (b) Did the straight-chain fatty acid contain an even or an odd number of methylene (OCH 2 O) groups (i.e., is n even or odd)? Explain. 12. Fatty Acid Oxidation in Uncontrolled Dia- betes When the acetyl-CoA produced during H9252 ox- idation in the liver exceeds the capacity of the citric acid cycle, the excess acetyl-CoA forms ketone bodies—acetone, acetoacetate, and D-H9252-hydroxybutyrate. This occurs in severe, uncontrolled diabetes: because the tissues cannot use glucose, they oxidize large amounts of fatty acids instead. Although acetyl-CoA is not toxic, the mitochondrion must divert the acetyl-CoA to ketone bodies. What problem would arise if acetyl-CoA were not converted to ketone bodies? How does the diversion to ketone bodies solve the problem? 13. Consequences of a High-Fat Diet with No Carbo- hydrates Suppose you had to subsist on a diet of whale blubber and seal blubber, with little or no carbohydrate. (a) What would be the effect of carbohydrate depriva- tion on the utilization of fats for energy? (b) If your diet were totally devoid of carbohydrate, would it be better to consume odd- or even-numbered fatty acids? Explain. 14. Metabolic Consequences of Ingesting H9275-Fluoro- oleate The shrub Dichapetalum toxicarium, native to Sierra Leone, produces H9275-fluorooleate, which is highly toxic to warm-blooded animals. This substance has been used as an arrow poison, and pow- dered fruit from the plant is sometimes used as a rat poison (hence the plant’s common name, ratsbane). Why is this sub- stance so toxic? (Hint: Review Chapter 16, Problem 13.) 15. Role of FAD as Electron Acceptor Acyl-CoA dehy- drogenase uses enzyme-bound FAD as a prosthetic group to dehydrogenate the H9251 and H9252 carbons of fatty acyl–CoA. What is the advantage of using FAD as an electron acceptor rather than NAD H11001 ? Explain in terms of the standard reduction po- tentials for the Enz-FAD/FADH 2 (EH11032H11034 H11005 H110020.219 V) and NAD H11001 /NADH (EH11032H11034 H11005 H110020.320 V) half-reactions. 16. H9252 Oxidation of Arachidic Acid How many turns of the fatty acid oxidation cycle are required for complete oxi- dation of arachidic acid (see Table 10–1) to acetyl-CoA? 17. Fate of Labeled Propionate If [3- 14 C]propionate ( 14 C in the methyl group) is added to a liver homogenate, 14 C-labeled oxaloacetate is rapidly produced. Draw a flow chart for the pathway by which propionate is transformed to oxaloacetate, and indicate the location of the 14 C in oxaloacetate. 18. Sources of H 2 O Produced in H9252 Oxidation The com- plete oxidation of palmitoyl-CoA to carbon dioxide and wa- ter is represented by the overall equation Palmitoyl-CoA H11001 23O 2 H11001 108P i H11001 108ADP On CoA H11001 16CO 2 H11001 108ATP H11001 23H 2 O Water is also produced in the reaction ADP H11001 P i O n ATP H11001 H 2 O but is not included as a product in the overall equation. Why? 19. Biological Importance of Cobalt In cattle, deer, sheep, and other ruminant animals, large amounts of pro- pionate are produced in the rumen through the bacterial fermentation of ingested plant matter. Propionate is the principal source of glucose for these animals, via the route propionate n oxaloacetate n glucose. In some areas of the world, notably Australia, ruminant animals sometimes show symptoms of anemia with concomitant loss of appetite and retarded growth, resulting from an inability to trans- form propionate to oxaloacetate. This condition is due to a cobalt deficiency caused by very low cobalt levels in the soil and thus in plant matter. Explain. 20. Fat Loss during Hibernation Bears expend about 25 H11003 10 6 J/day during periods of hibernation, which may last as long as seven months. The energy required to sustain life is obtained from fatty acid oxidation. How much weight loss (in kilograms) has occurred after seven months? How might ketosis be minimized during hibernation? (Assume the oxi- dation of fat yields 38 kJ/g.) (CH 2 ) 7 (CH 2 ) 7 H9275-Fluorooleate F CH 2 CC HH COO H11002 COO H11002 (CH 2 ) n CH 2 656 chapter W e now turn our attention to the amino acids, the fi- nal class of biomolecules that, through their oxida- tive degradation, make a significant contribution to the generation of metabolic energy. The fraction of meta- bolic energy obtained from amino acids, whether they are derived from dietary protein or from tissue protein, varies greatly with the type of organism and with meta- bolic conditions. Carnivores can obtain (immediately fol- lowing a meal) up to 90% of their energy requirements from amino acid oxidation, whereas herbivores may fill only a small fraction of their energy needs by this route. Most microorganisms can scavenge amino acids from their environment and use them as fuel when required by metabolic conditions. Plants, however, rarely if ever oxidize amino acids to provide energy; the carbohydrate produced from CO 2 and H 2 O in photosynthesis is gen- erally their sole energy source. Amino acid concentra- tions in plant tissues are carefully regulated to just meet the requirements for biosynthesis of proteins, nucleic acids, and other molecules needed to support growth. Amino acid catabolism does occur in plants, but its pur- pose is to produce metabolites for other biosynthetic pathways. In animals, amino acids undergo oxidative degrada- tion in three different metabolic circumstances: 1. During the normal synthesis and degradation of cellular proteins (protein turnover; Chapter 27), some amino acids that are released from protein breakdown and are not needed for new protein synthesis undergo oxidative degradation. 2. When a diet is rich in protein and the ingested amino acids exceed the body’s needs for protein synthesis, the surplus is catabolized; amino acids cannot be stored. 3. During starvation or in uncontrolled diabetes mel- litus, when carbohydrates are either unavailable or not properly utilized, cellular proteins are used as fuel. Under all these metabolic conditions, amino acids lose their amino groups to form H9251-keto acids, the “carbon skeletons” of amino acids. The H9251-keto acids undergo ox- idation to CO 2 and H 2 O or, often more importantly, pro- vide three- and four-carbon units that can be converted by gluconeogenesis into glucose, the fuel for brain, skeletal muscle, and other tissues. The pathways of amino acid catabolism are quite similar in most organisms. The focus of this chapter is on the pathways in vertebrates, because these have re- ceived the most research attention. As in carbohydrate and fatty acid catabolism, the processes of amino acid degradation converge on the central catabolic pathways, with the carbon skeletons of most amino acids finding their way to the citric acid cycle. In some cases the re- action pathways of amino acid breakdown closely par- allel steps in the catabolism of fatty acids (Chapter 17). AMINO ACID OXIDATION AND THE PRODUCTION OF UREA 18.1 Metabolic Fates of Amino Groups 657 18.2 Nitrogen Excretion and the Urea Cycle 665 18.3 Pathways of Amino Acid Degradation 671 I chose the study of the synthesis of urea in the liver because it appeared to be a relatively simple problem. —Hans Krebs, article in Perspectives in Biology and Medicine, 1970 18 8885d_c18_656-689 2/3/04 11:39 AM Page 656 mac76 mac76:385_reb: One important feature distinguishes amino acid degradation from other catabolic processes described to this point: every amino acid contains an amino group, and the pathways for amino acid degradation therefore include a key step in which the H9251-amino group is sepa- rated from the carbon skeleton and shunted into the pathways of amino group metabolism (Fig. 18–1). We deal first with amino group metabolism and nitrogen excretion, then with the fate of the carbon skeletons derived from the amino acids; along the way we see how the pathways are interconnected. 18.1 Metabolic Fates of Amino Groups Nitrogen, N 2 , is abundant in the atmosphere but is too inert for use in most biochemical processes. Because only a few microorganisms can convert N 2 to biologi- cally useful forms such as NH 3 (Chapter 22), amino groups are carefully husbanded in biological systems. Figure 18–2a provides an overview of the catabolic pathways of ammonia and amino groups in vertebrates. Amino acids derived from dietary protein are the source of most amino groups. Most amino acids are metabo- lized in the liver. Some of the ammonia generated in this process is recycled and used in a variety of biosynthetic pathways; the excess is either excreted directly or con- verted to urea or uric acid for excretion, depending on the organism (Fig. 18–2b). Excess ammonia generated in other (extrahepatic) tissues travels to the liver (in the form of amino groups, as described below) for con- version to the excretory form. Glutamate and glutamine play especially critical roles in nitrogen metabolism, acting as a kind of general collection point for amino groups. In the cytosol of hepatocytes, amino groups from most amino acids are transferred to H9251-ketoglutarate to form glutamate, which enters mitochondria and gives up its amino group to form NH 4 H11001 . Excess ammonia generated in most other tis- sues is converted to the amide nitrogen of glutamine, which passes to the liver, then into liver mitochondria. Glutamine or glutamate or both are present in higher concentrations than other amino acids in most tissues. In skeletal muscle, excess amino groups are gener- ally transferred to pyruvate to form alanine, another im- portant molecule in the transport of amino groups to the liver. We begin with a discussion of the breakdown of di- etary proteins, then give a general description of the metabolic fates of amino groups. 18.1 Metabolic Fates of Amino Groups 657 Intracellular protein Dietary protein Biosynthesis of amino acids, nucleotides, and biological amines Carbamoyl phosphate NH 4 H11001 CO 2 H11001 H 2 O H11001 ATP OxaloacetateUrea (nitrogen excretion product) Glucose (synthesized in gluconeogenesis) Carbon skeletons -Keto acids H9251 Amino acids Aspartate- arginino- succinate shunt of citric acid cycle Citric acid cycle Urea cycle FIGURE 18–1 Overview of amino acid catabolism in mammals. The amino groups and the carbon skeleton take separate but intercon- nected pathways. 8885d_c18_656-689 2/3/04 11:39 AM Page 657 mac76 mac76:385_reb: Dietary Protein Is Enzymatically Degraded to Amino Acids In humans, the degradation of ingested proteins to their constituent amino acids occurs in the gastrointestinal tract. Entry of dietary protein into the stomach stimu- lates the gastric mucosa to secrete the hormone gastrin, which in turn stimulates the secretion of hydrochloric acid by the parietal cells and pepsinogen by the chief cells of the gastric glands (Fig. 18–3a). The acidic gas- tric juice (pH 1.0 to 2.5) is both an antiseptic, killing most bacteria and other foreign cells, and a denaturing agent, unfolding globular proteins and rendering their internal peptide bonds more accessible to enzymatic hydrolysis. Pepsinogen (M r 40,554), an inactive precur- sor, or zymogen (p. 231), is converted to active pepsin (M r 34,614) by the enzymatic action of pepsin itself. In the stomach, pepsin hydrolyzes ingested proteins at pep- tide bonds on the amino-terminal side of the aromatic amino acid residues Phe, Trp, and Tyr (see Table 3–7), cleaving long polypeptide chains into a mixture of smaller peptides. As the acidic stomach contents pass into the small intestine, the low pH triggers secretion of the hormone secretin into the blood. Secretin stimulates the pan- creas to secrete bicarbonate into the small intestine to neutralize the gastric HCl, abruptly increasing the pH to about 7. (All pancreatic secretions pass into the small intestine through the pancreatic duct.) The digestion of proteins now continues in the small intestine. Arrival of amino acids in the upper part of the intestine (duode- num) causes release into the blood of the hormone Chapter 18 Amino Acid Oxidation and the Production of Urea658 O C Uric acid HN H N N H C C C O C O N H Uricotelic animals: birds, reptiles H 2 N NH 2 Urea O C Ureotelic animals: many terrestrial vertebrates; also sharks Ammonia (as ammonium ion) NH 4 H11001 Ammonotelic animals: most aquatic vertebrates, such as bony fishes and the larvae of amphibia Cellular protein COO H11002 C COO H11002 COO H11002 CH 2 CH 2 CO COO H11002 CH 3 CO O OH R H 3 N H11001 COO H11002 CHH 3 N H11001 NH 4 H11001 NH 2 Amino acids COO H11002 C R H9251-Keto acids H9251-Ketoglutarate H9251-Ketoglutarate COO H11002 CH 2 COO H11002 CHH 3 N H11001 CH 3 CH 2 COO H11002 C C HH 3 N H11001 CH 2 CH 2 Glutamate Glutamine Pyruvate Liver Alanine from muscle Glutamine from muscle and other tissues Amino acids from ingested protein NH 4 H11001 , urea, or uric acid FIGURE 18–2 Amino group catabolism. (a) Overview of catabolism of amino groups (shaded) in vertebrate liver. (b) Excretory forms of ni- trogen. Excess NH 4 H11001 is excreted as ammonia (microbes, bony fishes), urea (most terrestrial vertebrates), or uric acid (birds and terrestrial rep- tiles). Notice that the carbon atoms of urea and uric acid are highly oxidized; the organism discards carbon only after extracting most of its available energy of oxidation. (a) (b) 8885d_c18_656-689 2/3/04 11:39 AM Page 658 mac76 mac76:385_reb: cholecystokinin, which stimulates secretion of several pancreatic enzymes with activity optima at pH 7 to 8. Trypsinogen, chymotrypsinogen, and procarboxy- peptidases A and B, the zymogens of trypsin, chymo- trypsin, and carboxypeptidases A and B, are synthe- sized and secreted by the exocrine cells of the pancreas (Fig. 18–3b). Trypsinogen is converted to its active form, trypsin, by enteropeptidase, a proteolytic enzyme se- creted by intestinal cells. Free trypsin then catalyzes the conversion of additional trypsinogen to trypsin (see Fig. 6–33). Trypsin also activates chymotrypsinogen, the pro- carboxypeptidases, and proelastase. Why this elaborate mechanism for getting active di- gestive enzymes into the gastrointestinal tract? Synthe- sis of the enzymes as inactive precursors protects the exocrine cells from destructive proteolytic attack. The pancreas further protects itself against self-digestion by making a specific inhibitor, a protein called pancreatic trypsin inhibitor (p. 231), that effectively prevents premature production of active proteolytic enzymes within the pancreatic cells. Trypsin and chymotrypsin further hydrolyze the peptides that were produced by pepsin in the stom- ach. This stage of protein digestion is accomplished very efficiently, because pepsin, trypsin, and chymo- trypsin have different amino acid specificities (see Table 3–7). Degradation of the short peptides in the small intestine is then completed by other intestinal peptidases. These include carboxypeptidases A and B (both of which are zinc-containing enzymes), which remove successive carboxyl-terminal residues from peptides, and an aminopeptidase that hydrolyzes successive amino-terminal residues from short pep- tides. The resulting mixture of free amino acids is transported into the epithelial cells lining the small in- testine (Fig. 18–3c), through which the amino acids enter the blood capillaries in the villi and travel to the liver. In humans, most globular proteins from animal 18.1 Metabolic Fates of Amino Groups 659 Stomach (a) Gastric glands in stomach lining (b) Exocrine cells of pancreas (c) Villi of small intestine Pancreas Pancreatic duct Small intestine Low pH Pepsinogen pH 7 Zymogens active proteases Parietal cells (secrete HCl) Chief cells (secrete pepsinogen) Gastric mucosa (secretes gastrin) Rough ER Zymogen granules Collecting duct Villus Intestinal mucosa (absorbs amino acids) pepsin FIGURE 18–3 Part of the human digestive (gastrointestinal) tract. (a) The parietal cells and chief cells of the gastric glands secrete their products in response to the hormone gastrin. Pepsin begins the process of protein degradation in the stomach. (b) The cytoplasm of exocrine cells is completely filled with rough endoplasmic reticulum, the site of synthesis of the zymogens of many digestive enzymes. The zymogens are concentrated in membrane-enclosed transport particles called zymogen granules. When an exocrine cell is stimulated, its plasma membrane fuses with the zymogen granule membrane and zymogens are released into the lumen of the collecting duct by exocytosis. The collecting ducts ultimately lead to the pancreatic duct and thence to the small intestine. (c) Amino acids are absorbed through the epithelial cell layer (intestinal mucosa) of the villi and enter the capillaries. Recall that the products of lipid hydrolysis in the small intestine enter the lymphatic system after their absorption by the intestinal mucosa (see Fig. 17–1). 8885d_c18_656-689 2/3/04 11:39 AM Page 659 mac76 mac76:385_reb: sources are almost completely hydrolyzed to amino acids in the gastrointestinal tract, but some fibrous proteins, such as keratin, are only partly digested. In addition, the protein content of some plant foods is protected against breakdown by indigestible cellulose husks. Acute pancreatitis is a disease caused by ob- struction of the normal pathway by which pan- creatic secretions enter the intestine. The zymogens of the proteolytic enzymes are converted to their catalyt- ically active forms prematurely, inside the pancreatic cells, and attack the pancreatic tissue itself. This causes excruciating pain and damage to the organ that can prove fatal. ■ Pyridoxal Phosphate Participates in the Transfer of H9251-Amino Groups to H9251-Ketoglutarate The first step in the catabolism of most L-amino acids, once they have reached the liver, is removal of the H9251- amino groups, promoted by enzymes called amino- transferases or transaminases. In these transami- nation reactions, the H9251-amino group is transferred to the H9251-carbon atom of H9251-ketoglutarate, leaving behind the corresponding H9251-keto acid analog of the amino acid (Fig. 18–4). There is no net deamination (loss of amino groups) in these reactions, because the H9251-ketoglutarate becomes aminated as the H9251-amino acid is deaminated. The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of L-glutamate. The glutamate then functions as an amino group donor for biosynthetic pathways or for excretion pathways that lead to the elimination of nitrogenous waste products. Cells contain different types of aminotransferases. Many are specific for H9251-ketoglutarate as the amino group acceptor but differ in their specificity for the L-amino acid. The enzymes are named for the amino group donor (alanine aminotransferase, aspartate aminotransferase, for example). The reactions catalyzed by aminotrans- ferases are freely reversible, having an equilibrium con- stant of about 1.0 (H9004GH11032H11034 H11015 0 kJ/mol). All aminotransferases have the same prosthetic group and the same reaction mechanism. The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B 6 . We encountered pyridoxal phosphate in Chapter 15, as a coenzyme in the glycogen phosphorylase reaction, but its role in that reaction is not representative of its usual coenzyme function. Its primary role in cells is in the metabolism of molecules with amino groups. Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of amino- transferases. It undergoes reversible transformations between its aldehyde form, pyridoxal phosphate, which can accept an amino group, and its aminated form, pyri- doxamine phosphate, which can donate its amino group to an H9251-keto acid (Fig. 18–5a). Pyridoxal phosphate is generally covalently bound to the enzyme’s active site through an aldimine (Schiff base) linkage to the H9255-amino group of a Lys residue (Fig. 18–5b, d). Pyridoxal phosphate participates in a variety of re- actions at the H9251, H9252, and H9253 carbons (C-2 to C-4) of amino acids. Reactions at the H9251 carbon (Fig. 18–6) include racemizations (interconverting L- and D-amino acids) and decarboxylations, as well as transaminations. Pyri- doxal phosphate plays the same chemical role in each of these reactions. A bond to the H9251 carbon of the sub- strate is broken, removing either a proton or a carboxyl group. The electron pair left behind on the H9251 carbon would form a highly unstable carbanion, but pyridoxal phosphate provides resonance stabilization of this in- termediate (Fig. 18–6 inset). The highly conjugated structure of PLP (an electron sink) permits delocaliza- tion of the negative charge. Aminotransferases (Fig. 18–5) are classic examples of enzymes catalyzing bimolecular Ping-Pong reactions (see Fig. 6–13b), in which the first substrate reacts and the product must leave the active site before the sec- ond substrate can bind. Thus the incoming amino acid binds to the active site, donates its amino group to pyri- doxal phosphate, and departs in the form of an H9251-keto acid. The incoming H9251-keto acid then binds, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid. As described in Box 18–1 on page 664, measurement of the alanine aminotrans- ferase and aspartate aminotransferase levels in blood serum is important in some medical diagnoses. Chapter 18 Amino Acid Oxidation and the Production of Urea660 O HH 3 N H11001 COO H11002 C COO H11002 CH 2 CH 2 CO R COO H11002 amino- transferase H 3 N H11001 C COO H11002 R COO H11002 CH 2 CH 2 HC COO H11002 H9251-Keto acid L-Glutamate L-Amino acid H9251-Ketoglutarate PLP FIGURE 18–4 Enzyme-catalyzed transaminations. In many amino- transferase reactions, H9251-ketoglutarate is the amino group acceptor. All aminotransferases have pyridoxal phosphate (PLP) as cofactor. Al- though the reaction is shown here in the direction of transfer of the amino group to H9251-ketoglutarate, it is readily reversible. 8885d_c18_656-689 2/3/04 11:39 AM Page 660 mac76 mac76:385_reb: Glutamate Releases Its Amino Group as Ammonia in the Liver As we have seen, the amino groups from many of the H9251-amino acids are collected in the liver in the form of the amino group of L-glutamate molecules. These amino groups must next be removed from glutamate to pre- pare them for excretion. In hepatocytes, glutamate is transported from the cytosol into mitochondria, where it undergoes oxidative deamination catalyzed by L- glutamate dehydrogenase (M r 330,000). In mammals, this enzyme is present in the mitochondrial matrix. It is the only enzyme that can use either NAD H11001 or NADP H11001 as the acceptor of reducing equivalents (Fig. 18–7). The combined action of an aminotransferase and glutamate dehydrogenase is referred to as transdeam- ination. A few amino acids bypass the transdeamina- tion pathway and undergo direct oxidative deamination. The fate of the NH 4 H11001 produced by any of these deami- nation processes is discussed in detail in Section 18.2. The H9251-ketoglutarate formed from glutamate deamina- tion can be used in the citric acid cycle and for glucose synthesis. Glutamate dehydrogenase operates at an important intersection of carbon and nitrogen metabolism. An al- losteric enzyme with six identical subunits, its activity is influenced by a complicated array of allosteric mod- ulators. The best-studied of these are the positive mod- ulator ADP and the negative modulator GTP. The meta- bolic rationale for this regulatory pattern has not been elucidated in detail. Mutations that alter the allosteric binding site for GTP or otherwise cause permanent acti- vation of glutamate dehydrogenase lead to a human ge- netic disorder called hyperinsulinism-hyperammonemia 18.1 Metabolic Fates of Amino Groups 661 FIGURE 18–5 Pyridoxal phosphate, the prosthetic group of amino- transferases. (a) Pyridoxal phosphate (PLP) and its aminated form, pyri- doxamine phosphate, are the tightly bound coenzymes of amino- transferases. The functional groups are shaded. (b) Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiff- base linkage to a Lys residue at the active site. The steps in the for- mation of a Schiff base from a primary amine and a carbonyl group are detailed in Figure 14–5. (c) PLP (red) bound to one of the two ac- tive sites of the dimeric enzyme aspartate aminotransferase, a typical aminotransferase; (d) close-up view of the active site, with PLP (red, with yellow phosphorus) in aldimine linkage with the side chain of Lys 258 (purple); (e) another close-up view of the active site, with PLP linked to the substrate analog 2-methylaspartate (green) via a Schiff base (PDB ID 1AJS). (d) (c) (e) O H11002 H11002 O P N O OH CH 2 CH 3 O O H11002 H11002 O P C O OH CH 2 H CH 3 O O H (b) O H11002 H11002 O P C O OH CH 2 H H11001 CH 3 NH H 3 N O O H11002 H11002 O P C O OH CH 2 H CH 3 O O H Pyridoxal phosphate (PLP) Pyridoxamine phosphate (a) Enz Enz Lys NH 2 H 2 O Lys H C Schiff base H11001 H11001H11001 NH H11001 NH H11001 NH 8885d_c18_656-689 2/3/04 11:39 AM Page 661 mac76 mac76:385_reb: syndrome, characterized by elevated levels of ammonia in the bloodstream and hypoglycemia. Glutamine Transports Ammonia in the Bloodstream Ammonia is quite toxic to animal tissues (we examine some possible reasons for this toxicity later), and the levels present in blood are regulated. In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia. In most animals much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys. For this transport function, glutamate, critical to intra- cellular amino group metabolism, is supplanted by L-glutamine. The free ammonia produced in tissues is combined with glutamate to yield glutamine by the ac- tion of glutamine synthetase. This reaction requires Chapter 18 Amino Acid Oxidation and the Production of Urea662 R H CH N H P Amine H11001 N H P CH C H11001 NH H11001 CH 3 NH H11001 H C H H11002 phosphate H CH N H P H11001 C H11001 NH Quinonoid intermediate Resonance structures for stabili- zation of a carbanion by PLP Carbanion RR R H C H H11001 NH 3 R N H P H11001 CH NH H11001 H C COO H11002 H11001 R H C COO H11002 H11001 NH 3 Pyridoxal phosphate (aldimine form, on regenerated enzyme) Pyridoxal phosphate (aldimine form, on regenerated enzyme) H11001 H11001 Lys Enz NH 3 H11001 Lys Enz NH 3 CH 3 CH 3 CH CO 2 3 HO R CH 2 H9251-Keto acid C H11001 NH 3 Pyridoxamine O H11001 N H H11001 P COO H11002 CH 3 HO HO HO HO H N Lys CH N H P H11001 HO H11001 R H C H11001 H11001 NH 3 L-Amino acid Pyridoxal phosphate (aldimine form, on enzyme) COO H11002 CH 3 Enz H 2 O COO H11002 C CH 2 N H P H11001 NH CH 3 HO R H11001 D-Amino acid A B C Schiff base intermediate (aldimine) R C N H P H11001 CH NH H11001 H B C COO H11002 CH 3 HO : Schiff base intermediate (aldimine) R N H P H11001 CH NH H11001 H HC C O H11002 O CH 3 HO R N H P CH NH H11001 H H11001 H C COO H11002 CH 3 HO Quinonoid intermediate Quinonoid intermediate R CH COO H11002 N H P C H11001 NH H H11001 CH 3 HO : : R N H P C NH H11001 C CH 3 HO Quinonoid intermediate : MECHANISM FIGURE 18–6 Some amino acid transformations at the H9251 carbon that are facilitated by pyridoxal phosphate. Pyridoxal phos- phate is generally bonded to the enzyme through a Schiff base (see Fig. 18–5b, d). Reactions begin (top left) with formation of a new Schiff base (aldimine) between the H9251-amino group of the amino acid and PLP, which substitutes for the enzyme-PLP linkage. Three alternative fates for this Schiff base are shown: A transamination, B racemiza- tion, and C decarboxylation. The Schiff base formed between PLP and the amino acid is in conjugation with the pyridine ring, an electron sink that permits delocalization of an electron pair to avoid formation of an unstable carbanion on the H9251 carbon (inset). A quinonoid inter- mediate is involved in all three types of reactions. The transamination route ( A ) is especially important in the pathways described in this chapter. The pathway highlighted here (shown left to right) represents only part of the overall reaction catalyzed by aminotransferases. To complete the process, a second H9251-keto acid replaces the one that is released, and this is converted to an amino acid in a reversal of the reaction steps (right to left). Pyridoxal phosphate is also involved in certain reactions at the H9252 and H9253 carbons of some amino acids (not shown). Pyridoxal Phosphate Reaction Mechanisms 8885d_c18_656-689 2/3/04 11:39 AM Page 662 mac76 mac76:385_reb: ATP and occurs in two steps (Fig. 18–8). First, gluta- mate and ATP react to form ADP and a H9253-glutamyl phos- phate intermediate, which then reacts with ammonia to produce glutamine and inorganic phosphate. Glutamine is a nontoxic transport form of ammonia; it is normally present in blood in much higher concentrations than other amino acids. Glutamine also serves as a source of amino groups in a variety of biosynthetic reactions. Glu- tamine synthetase is found in all organisms, always play- ing a central metabolic role. In microorganisms, the en- zyme serves as an essential portal for the entry of fixed nitrogen into biological systems. (The roles of glutamine and glutamine synthetase in metabolism are further dis- cussed in Chapter 22.) In most terrestrial animals, glutamine in excess of that required for biosynthesis is transported in the blood to the intestine, liver, and kidneys for processing. In these tissues, the amide nitrogen is released as ammonium ion in the mitochondria, where the enzyme glutaminase converts glutamine to glutamate and NH 4 H11001 (Fig. 18–8). The NH 4 H11001 from intestine and kidney is transported in the blood to the liver. In the liver, the ammonia from all sources is disposed of by urea synthesis. Some of the glu- tamate produced in the glutaminase reaction may be fur- ther processed in the liver by glutamate dehydrogenase, releasing more ammonia and producing carbon skeletons for metabolic fuel. However, most glutamate enters the transamination reactions required for amino acid biosyn- thesis and other processes (Chapter 22). In metabolic acidosis (p. 652) there is an increase in glutamine processing by the kidneys. Not all the excess NH 4 H11001 thus produced is released into the bloodstream or converted to urea; some is excreted di- rectly into the urine. In the kidney, the NH 4 H11001 forms salts with metabolic acids, facilitating their removal in the urine. Bicarbonate produced by the decarboxylation of H9251-ketoglutarate in the citric acid cycle can also serve as a buffer in blood plasma. Taken together, these effects of glutamine metabolism in the kidney tend to counter- act acidosis. ■ 18.1 Metabolic Fates of Amino Groups 663 H H11001 COO H11002 C O -Ketoglutarate Glutamate COO H11002 CH 2 CH 2 COO H11002 COO H11002 C CH 2 NH 4 H11001 H 2 N H11001 H 2 O H 3 N CH 2 COO H11002 COO H11002 C CH 2 CH 2 NAD(P) H11001 NAD(P)H FIGURE 18–7 Reaction catalyzed by glutamate dehydrogenase. The glutamate dehydrogenase of mammalian liver has the unusual capac- ity to use either NAD H11001 or NADP H11001 as cofactor. The glutamate dehy- drogenases of plants and microorganisms are generally specific for one or the other. The mammalian enzyme is allosterically regulated by GTP and ADP. CCHCH 2 NH 3 COO H11002 CH 2 H11002 OOC CHCH 2 COO H11002 CH 2 O CCHCH 2 O H11002 H11001 O H11002 O COO H11002 CH 2 ATP ADP OP O glutamine synthetase glutaminase (liver mitochondria) L-Glutamine L-Glutamate L-Glutamate CCHCH 2 H 2 N COO H11002 CH 2 O NH 4 H11001 H11002 O P i glutamine synthetase H9253-Glutamyl phosphate NH 4 H11001 Urea H 2 O NH 3 H11001 NH 3 H11001 NH 3 H11001 FIGURE 18–8 Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. After transport in the bloodstream, the glutamine enters the liver and NH 4 H11001 is liberated in mitochondria by the enzyme glutaminase. 8885d_c18_656-689 2/3/04 11:39 AM Page 663 mac76 mac76:385_reb: Alanine Transports Ammonia from Skeletal Muscles to the Liver Alanine also plays a special role in transporting amino groups to the liver in a nontoxic form, via a pathway called the glucose-alanine cycle (Fig. 18–9). In mus- cle and certain other tissues that degrade amino acids for fuel, amino groups are collected in the form of glutamate by transamination (Fig. 18–2a). Glutamate can be converted to glutamine for transport to the liver, as described above, or it can transfer its H9251-amino group to pyruvate, a readily available product of muscle glycolysis, by the action of alanine aminotransferase (Fig. 18–9). The alanine so formed passes into the blood and travels to the liver. In the cytosol of hepatocytes, alanine aminotransferase transfers the amino group from alanine to H9251-ketoglutarate, forming pyruvate and glutamate. Glutamate can then enter mitochondria, where the glutamate dehydrogenase reaction releases NH 4 H11001 (Fig. 18–7), or can undergo transamination with oxaloacetate to form aspartate, another nitrogen donor in urea synthesis, as we shall see. The use of alanine to transport ammonia from skeletal muscles to the liver is another example of the intrinsic economy of living organisms. Vigorously con- tracting skeletal muscles operate anaerobically, produc- ing pyruvate and lactate from glycolysis as well as Chapter 18 Amino Acid Oxidation and the Production of Urea664 BOX 18–1 BIOCHEMISTRY IN MEDICINE Assays for Tissue Damage Analyses of certain enzyme activities in blood serum give valuable diagnostic information for a number of disease conditions. Alanine aminotransferase (ALT; also called glutamate-pyruvate transaminase, GPT) and aspar- tate aminotransferase (AST; also called glutamate- oxaloacetate transaminase, GOT) are important in the diagnosis of heart and liver damage caused by heart attack, drug toxicity, or infection. After a heart attack, a variety of enzymes, including these aminotrans- ferases, leak from the injured heart cells into the bloodstream. Measurements of the blood serum con- centrations of the two aminotransferases by the SGPT and SGOT tests (S for serum)—and of another en- zyme, creatine kinase, by the SCK test—can pro- vide information about the severity of the damage. Creatine kinase is the first heart enzyme to appear in the blood after a heart attack; it also disappears quickly from the blood. GOT is the next to appear, and GPT follows later. Lactate dehydrogenase also leaks from injured or anaerobic heart muscle. The SGOT and SGPT tests are also important in occupational medicine, to determine whether people exposed to carbon tetrachloride, chloroform, or other industrial solvents have suffered liver damage. Liver degeneration caused by these solvents is accompanied by leakage of various enzymes from injured hepato- cytes into the blood. Aminotransferases are most use- ful in the monitoring of people exposed to these chem- icals, because these enzyme activities are high in liver and can be detected in very small amounts. Blood glucose Glucose Glucose Pyruvate Pyruvate Blood alanine Alanine Alanine Glutamate Glutamate Amino acids Muscle protein Urea NH 4 H11001 NH 4 H11001 urea cycle alanine aminotransferase alanine aminotransferase gluconeo- genesis glycolysis -KetoglutarateH9251 -KetoglutarateH9251 Liver FIGURE 18–9 Glucose-alanine cycle. Alanine serves as a carrier of ammonia and of the carbon skeleton of pyruvate from skeletal mus- cle to liver. The ammonia is excreted and the pyruvate is used to pro- duce glucose, which is returned to the muscle. 8885d_c18_656-689 2/3/04 11:39 AM Page 664 mac76 mac76:385_reb: ammonia from protein breakdown. These products must find their way to the liver, where pyruvate and lactate are incorporated into glucose, which is returned to the muscles, and ammonia is converted to urea for excre- tion. The glucose-alanine cycle, in concert with the Cori cycle (see Box 14–1 and Fig. 23–18), accomplishes this transaction. The energetic burden of gluconeogenesis is thus imposed on the liver rather than the muscle, and all available ATP in muscle is devoted to muscle contraction. Ammonia Is Toxic to Animals The catabolic production of ammonia poses a se- rious biochemical problem, because ammonia is very toxic. The molecular basis for this toxicity is not entirely understood. The terminal stages of ammonia in- toxication in humans are characterized by onset of a comatose state accompanied by cerebral edema (an in- crease in the brain’s water content) and increased cra- nial pressure, so research and speculation on ammonia toxicity have focused on this tissue. Speculation centers on a potential depletion of ATP in brain cells. Ridding the cytosol of excess ammonia requires re- ductive amination of H9251-ketoglutarate to glutamate by glutamate dehydrogenase (the reverse of the reaction described earlier; Fig. 18–7) and conversion of gluta- mate to glutamine by glutamine synthetase. Both en- zymes are present at high levels in the brain, although the glutamine synthetase reaction is almost certainly the more important pathway for removal of ammonia. High levels of NH 4 H11001 lead to increased levels of glutamine, which acts as an osmotically active solute (osmolyte) in brain astrocytes, star-shaped cells of the nervous sys- tem that provide nutrients, support, and insulation for neurons. This triggers an uptake of water into the as- trocytes to maintain osmotic balance, leading to swelling and the symptoms noted above. Depletion of glutamate in the glutamine synthetase reaction may have additional effects on the brain. Glu- tamate and its derivative H9253-aminobutyrate (GABA; see Fig. 22–29) are important neurotransmitters; the sensi- tivity of the brain to ammonia may reflect a depletion of neurotransmitters as well as changes in cellular os- motic balance. ■ As we close this discussion of amino group metabolism, note that we have described several processes that de- posit excess ammonia in the mitochondria of hepatocytes (Fig. 18–2). We now look at the fate of that ammonia. SUMMARY 18.1 Metabolic Fates of Amino Groups ■ Humans derive a small fraction of their oxidative energy from the catabolism of amino acids. Amino acids are derived from the normal breakdown (recycling) of cellular proteins, degradation of ingested proteins, and breakdown of body proteins in lieu of other fuel sources during starvation or in uncontrolled diabetes mellitus. ■ Proteases degrade ingested proteins in the stomach and small intestine. Most proteases are initially synthesized as inactive zymogens. ■ An early step in the catabolism of amino acids is the separation of the amino group from the carbon skeleton. In most cases, the amino group is transferred to H9251-ketoglutarate to form glutamate. This transamination reaction requires the coenzyme pyridoxal phosphate. ■ Glutamate is transported to liver mitochondria, where glutamate dehydrogenase liberates the amino group as ammonium ion (NH 4 H11001 ). Ammonia formed in other tissues is transported to the liver as the amide nitrogen of glutamine or, in transport from skeletal muscle, as the amino group of alanine. ■ The pyruvate produced by deamination of alanine in the liver is converted to glucose, which is transported back to muscle as part of the glucose-alanine cycle. 18.2 Nitrogen Excretion and the Urea Cycle If not reused for the synthesis of new amino acids or other nitrogenous products, amino groups are chan- neled into a single excretory end product (Fig. 18–10). Most aquatic species, such as the bony fishes, are ammonotelic, excreting amino nitrogen as ammonia. The toxic ammonia is simply diluted in the surrounding water. Terrestrial animals require pathways for nitrogen excretion that minimize toxicity and water loss. Most terrestrial animals are ureotelic, excreting amino nitrogen in the form of urea; birds and reptiles are uricotelic, excreting amino nitrogen as uric acid. (The pathway of uric acid synthesis is described in Fig. 22–45.) Plants recycle virtually all amino groups, and nitrogen excretion occurs only under very unusual circumstances. In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes is converted to urea in the urea cycle. This pathway was discovered in 1932 by Hans Krebs (who later also discovered the citric acid cycle) and a medical student associate, Kurt Henseleit. Urea production occurs almost exclusively in the liver and is the fate of most of the ammonia channeled there. The urea passes into the bloodstream and thus to the kidneys and is excreted into the urine. The production of urea now becomes the focus of our discussion. 18.2 Nitrogen Excretion and the Urea Cycle 665 8885d_c18_656-689 2/3/04 11:39 AM Page 665 mac76 mac76:385_reb: Chapter 18 Amino Acid Oxidation and the Production of Urea666 glutamate dehydrogenase NH 4 Glutamine glutaminase carbamoyl phosphate synthetase I Alanine (from muscle) Mitochondrial matrix Cytosol -Keto- glutarate Oxaloacetate Aspartate aspartate aminotransferase 2 ATP Amino acids H11002 H11001 2ADP H11001 P i NH 3 H11001 CH COOR H11002 NH 3 H11001 CH COOCH 3 H11002 NH 3 H11001 CH COO H11002 CH 2 CH 2 OOC H11002 O C COOCH 2 H11002 OOC H11002 CH COOCH 2 H11002 OOC H11002 NH 3 H11001 NH 3 H11001 CH COOCH 2 H11002 CH 2 C O H 2 N Glutamate Glutamate Carbamoyl phosphate C O P O H11002 H 2 N O H11002 OO Glutamine (from extrahepatic tissues) -Ketoglutarate -Keto acid HCO 3 H 2 O Aspartate AMP Urea cycle 2b 3 4 P i CH COOCH 2 H11002 OOC H11002 NH 3 H11001 C N H CH COO H11002 H 2 N (CH 2 ) 3 NH 2 H11001 NH 3 H11001 Fumarate COO H11002 CHCHOOC H11002 H 3 N CH COO H11002 (CH 2 ) 3 NH 3 H11001 H11001 Urea CH 2 N O NH 2 Citrullyl-AMP intermediate NH 3 NH 2 H11001 CH COO(CH 2 ) 3 CH 2 H11002 NHC O O O H OH OH HH N N N N H P HN O H11002 O 2a PP i ATP Ornithine Citrulline Arginine C NH CH COO H11002 NH (CH 2 ) 3 NH 2 H11001 NH 3 H11001 CHCH 2 OOC H11002 COO H11002 Argininosuccinate Ornithine C CH COO H11002 H 2 N NH 3 H11001 O Citrulline NH (CH 2 ) 3 1 8885d_c18_656-689 2/3/04 11:39 AM Page 666 mac76 mac76:385_reb: Urea Is Produced from Ammonia in Five Enzymatic Steps The urea cycle begins inside liver mitochondria, but three of the subsequent steps take place in the cytosol; the cycle thus spans two cellular compartments (Fig. 18–10). The first amino group to enter the urea cycle is derived from ammonia in the mitochondrial matrix—NH 4 H11001 arising by the pathways described above. The liver also receives some ammonia via the portal vein from the intestine, from the bacterial oxidation of amino acids. Whatever its source, the NH 4 H11001 generated in liver mitochondria is immediately used, together with CO 2 (as HCO 3 H11002 ) produced by mitochondrial respiration, to form carbamoyl phosphate in the matrix (Fig. 18–11a; see also Fig. 18–10). This ATP-dependent reaction is catalyzed by carbamoyl phosphate synthetase I, a regulatory enzyme (see below). The mitochondrial form of the enzyme is distinct from the cytosolic (II) form, which has a separate function in pyrimidine biosynthe- sis (Chapter 22). The carbamoyl phosphate, which functions as an ac- tivated carbamoyl group donor, now enters the urea cy- cle. The cycle has four enzymatic steps. First, carbamoyl phosphate donates its carbamoyl group to ornithine to form citrulline, with the release of P i (Fig. 18–10, step 1 ). Ornithine plays a role resembling that of oxaloac- etate in the citric acid cycle, accepting material at each turn of the cycle. The reaction is catalyzed by ornithine transcarbamoylase, and the citrulline passes from the mitochondrion to the cytosol. The second amino group now enters from aspartate (generated in mitochondria by transamination and trans- ported into the cytosol) by a condensation reaction between the amino group of aspartate and the ureido 18.2 Nitrogen Excretion and the Urea Cycle 667 FIGURE 18–10 (facing page) Urea cycle and reactions that feed amino groups into the cycle. The enzymes catalyzing these reactions (named in the text) are distributed between the mitochondrial matrix and the cytosol. One amino group enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, formed in the matrix by transamination of oxaloacetate and glutamate, cat- alyzed by aspartate aminotransferase. The urea cycle consists of four steps. 1 Formation of citrulline from ornithine and carbamoyl phos- phate (entry of the first amino group); the citrulline passes into the cy- tosol. 2 Formation of argininosuccinate through a citrullyl-AMP in- termediate (entry of the second amino group). 3 Formation of arginine from argininosuccinate; this reaction releases fumarate, which enters the citric acid cycle. 4 Formation of urea; this reaction also regen- erates, ornithine. The pathways by which NH 4 H11001 arrives in the mito- chondrial matrix of hepatocytes were discussed in Section 18.1. 1 O C OH OH NH 3 – O O C ATP Bicarbonate Carbonic-phosphoric acid anhydride Carbamoyl phosphate Carbamate ADP ADP 2 P i 3 ADP O – O P O – O O – O P O – O – O O P O – O ATP : : O – O CH 2 N O CH 2 N : 1 PP i AMP AMP Aspartate 2 ATP Citrulline Citrullyl-AMP Argininosuccinate Adenosine O O P O – O O P O – O O – P O – O : NH 2 NH 3 + NH : CO (CH 2 ) 3 COO – CH + NH 2 H 2 N NH 3 + + NH : CO (CH 2 ) 3 COO – CH NH 2 NH 3 + + NH CCNH (CH 2 ) 3 COO – COO – COO – CH 2 CH COO – CH 2 C H COO – H MECHANISM FIGURE 18–11 Nitrogen-acquiring reactions in the syn- thesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (a) In the reaction catalyzed by carbamoyl phosphate synthetase I, the first nitrogen enters from ammonia. The terminal phos- phate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. In other words, this reaction has two activa- tion steps ( 1 and 3 ). Carbamoyl Phosphate Synthetase I Mech- anism (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. The ureido oxygen of citrulline is activated by the addition of AMP in step 1 ; this sets up the addi- tion of aspartate in step 2 , with AMP (including the ureido oxygen) as the leaving group. Argininosuccinate Synthetase Mechanism (a) (b) 8885d_c18_667 2/3/04 4:13 PM Page 667 mac76 mac76:385_reb: (carbonyl) group of citrulline, forming argininosucci- nate (step 2 in Fig. 18–10). This cytosolic reaction, cat- alyzed by argininosuccinate synthetase, requires ATP and proceeds through a citrullyl-AMP intermediate (Fig. 18–11b). The argininosuccinate is then cleaved by argininosuccinase (step 3 in Fig. 18–10) to form free arginine and fumarate, the latter entering mitochondria to join the pool of citric acid cycle intermediates. This is the only reversible step in the urea cycle. In the last reaction of the urea cycle (step 4 ), the cytosolic en- zyme arginase cleaves arginine to yield urea and or- nithine. Ornithine is transported into the mitochondrion to initiate another round of the urea cycle. As we noted in Chapter 16, the enzymes of many metabolic pathways are clustered (p. 605), with the product of one enzyme reaction being channeled di- rectly to the next enzyme in the pathway. In the urea cycle, the mitochondrial and cytosolic enzymes appear to be clustered in this way. The citrulline transported out of the mitochondrion is not diluted into the general pool of metabolites in the cytosol but is passed directly to the active site of argininosuccinate synthetase. This channeling between enzymes continues for argini- nosuccinate, arginine, and ornithine. Only urea is re- leased into the general cytosolic pool of metabolites. The Citric Acid and Urea Cycles Can Be Linked Because the fumarate produced in the argininosucci- nase reaction is also an intermediate of the citric acid cycle, the cycles are, in principle, interconnected—in a process dubbed the “Krebs bicycle” (Fig. 18–12). How- ever, each cycle can operate independently and com- munication between them depends on the transport of key intermediates between the mitochondrion and cy- tosol. Several enzymes of the citric acid cycle, includ- ing fumarase (fumarate hydratase) and malate dehy- drogenase (p. 612), are also present as isozymes in the cytosol. The fumarate generated in cytosolic arginine synthesis can therefore be converted to malate in the cytosol, and these intermediates can be further metab- olized in the cytosol or transported into mitochondria for use in the citric acid cycle. Aspartate formed in mitochondria by transamination between oxaloacetate and glutamate can be transported to the cytosol, where it serves as nitrogen donor in the urea cycle reaction catalyzed by argininosuccinate synthetase. These reac- tions, making up the aspartate-argininosuccinate shunt, provide metabolic links between the separate pathways by which the amino groups and carbon skele- tons of amino acids are processed. Chapter 18 Amino Acid Oxidation and the Production of Urea668 Mitochondrial matrix Cytosol Ornithine Ornithine Urea ArginineFumarate Malate Urea cycle Aspartate-argininosuccinate shunt of citric acid cycle Citrulline Carbamoyl phosphate Citrulline Aspartate Aspartate Glutamate a-Ketoglutarate NADH NAD H11001 Citric acid cycle Fumarate Malate Arginino- succinate FIGURE 18–12 Links between the urea cycle and citric acid cycle. The interconnected cycles have been called the “Krebs bicycle.” The pathways linking the citric acid and urea cycles are called the aspartate-argininosuccinate shunt; these effectively link the fates of the amino groups and the carbon skeletons of amino acids. The inter- connections are even more elaborate than the arrows suggest. For example, some citric acid cycle enzymes, such as fumarase and malate dehydrogenase, have both cytosolic and mitochondrial isozymes. Fu- marate produced in the cytosol—whether by the urea cycle, purine biosynthesis, or other processes—can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria (via the malate-aspartate shuttle; see Fig. 19–27) to enter the citric acid cycle. 8885d_c18_656-689 2/3/04 11:39 AM Page 668 mac76 mac76:385_reb: The Activity of the Urea Cycle Is Regulated at Two Levels The flux of nitrogen through the urea cycle in an indi- vidual animal varies with diet. When the dietary intake is primarily protein, the carbon skeletons of amino acids are used for fuel, producing much urea from the excess amino groups. During prolonged starvation, when break- down of muscle protein begins to supply much of the organism’s metabolic energy, urea production also in- creases substantially. These changes in demand for urea cycle activity are met over the long term by regulation of the rates of syn- thesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver. All five enzymes are synthesized at higher rates in starving animals and in animals on very-high-protein diets than in well-fed ani- mals eating primarily carbohydrates and fats. Animals on protein-free diets produce lower levels of urea cycle enzymes. On a shorter time scale, allosteric regulation of at least one key enzyme adjusts the flux through the urea cycle. The first enzyme in the pathway, carbamoyl phosphate synthetase I, is allosterically activated by N-acetylglutamate, which is synthesized from acetyl- CoA and glutamate by N-acetylglutamate synthase (Fig. 18–13). In plants and microorganisms this enzyme catalyzes the first step in the de novo synthesis of argi- nine from glutamate (see Fig. 22–10), but in mammals N-acetylglutamate synthase activity in the liver has a purely regulatory function (mammals lack the other en- zymes needed to convert glutamate to arginine). The steady-state levels of N-acetylglutamate are determined by the concentrations of glutamate and acetyl-CoA (the substrates for N-acetylglutamate synthase) and arginine (an activator of N-acetylglutamate synthase, and thus an activator of the urea cycle). Pathway Interconnections Reduce the Energetic Cost of Urea Synthesis If we consider the urea cycle in isolation, we see that the synthesis of one molecule of urea requires four high- energy phosphate groups (Fig. 18–10). Two ATP mole- cules are required to make carbamoyl phosphate, and one ATP to make argininosuccinate—the latter ATP un- dergoing a pyrophosphate cleavage to AMP and PP i , which is hydrolyzed to two P i . The overall equation of the urea cycle is 2NH 4 H11001 H11001 HCO 3 H11002 H11001 3ATP 4H11002 H11001 H 2 O 88n urea H11001 2ADP 3H11002 H11001 4P i 2H11002 H11001 AMP 2H11002 H11001 2H H11001 However, the urea cycle also causes a net conversion of oxaloacetate to fumarate (via aspartate), and the re- generation of oxaloacetate (Fig. 18–12) produces NADH in the malate dehydrogenase reaction. Each NADH mol- ecule can generate up to 2.5 ATP during mitochondrial respiration (Chapter 19), greatly reducing the overall energetic cost of urea synthesis. Genetic Defects in the Urea Cycle Can Be Life-Threatening People with genetic defects in any enzyme in- volved in urea formation cannot tolerate protein- rich diets. Amino acids ingested in excess of the mini- mum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea and exported into the bloodstream, and, as we have seen, ammonia is highly toxic. The absence of a urea cycle enzyme can result in hyperammonemia or in the build-up of one or more urea cycle intermediates, depending on the enzyme that is missing. Given that most urea cycle steps are irre- versible, the absent enzyme activity can often be iden- tified by determining which cycle intermediate is pres- ent in especially elevated concentration in the blood and/or urine. Although the breakdown of amino acids can have serious health consequences in individuals with urea cycle deficiencies, a protein-free diet is not a treatment option. Humans are incapable of synthesizing half of the 20 common amino acids, and these essential amino acids (Table 18–1) must be provided in the diet. 18.2 Nitrogen Excretion and the Urea Cycle 669 H11001 Carbamoyl phosphate CH 2 CH 2 S-CoA H11001 COO H11002 C COO H11002 Acetyl-CoA N-Acetylglutamate N-acetylglutamate synthase CH 3 O C HC CH 2 O H11002 H C P CH 3 H11001 CH 2 CoA-SH Glutamate Arginine H11001 H 2 N COO H11002 HCO 3 H11002 NH 4 2ATP H 3 N NHC O O O O COO H11002 O H11002 2ADP H11001 P i carbamoyl phosphate synthetase I FIGURE 18–13 Synthesis of N-acetylglutamate and its activation of carbamoyl phosphate synthetase I. 8885d_c18_656-689 2/3/04 11:39 AM Page 669 mac76 mac76:385_reb: A variety of treatments are available for individuals with urea cycle defects. Careful administration of the aro- matic acids benzoate or phenylbutyrate in the diet can help lower the level of ammonia in the blood. Benzoate is converted to benzoyl-CoA, which combines with glycine to form hippurate (Fig. 18–14, left). The glycine used up in this reaction must be regenerated, and ammonia is thus taken up in the glycine synthase reac- tion. Phenylbutyrate is converted to phenylacetate by H9252 oxidation. The phenylacetate is then converted to phenylacetyl-CoA, which combines with glutamine to form phenylacetylglutamine (Fig. 18–14, right). The re- sulting removal of glutamine triggers its further synthe- sis by glutamine synthetase (see Eqn 22–1) in a reaction that takes up ammonia. Both hippurate and phenylacetyl- glutamine are nontoxic compounds that are excreted in the urine. The pathways shown in Figure 18–14 make only minor contributions to normal metabolism, but they become prominent when aromatic acids are ingested. Other therapies are more specific to a particular en- zyme deficiency. Deficiency of N-acetylglutamate syn- thase results in the absence of the normal activator of carbamoyl phosphate synthetase I (Fig. 18–13). This condition can be treated by administering carbamoyl glutamate, an analog of N-acetylglutamate that is effec- tive in activating carbamoyl phosphate synthetase I. Supplementing the diet with arginine is useful in treat- ing deficiencies of ornithine transcarbamoylase, argini- nosuccinate synthetase, and argininosuccinase. Many NH Carbamoyl glutamate H 2 N CH 2 CH 2 CHC O COO H11002 COO H11002 of these treatments must be accompanied by strict di- etary control and supplements of essential amino acids. In the rare cases of arginase deficiency, arginine, the substrate of the defective enzyme, must be excluded from the diet. ■ Chapter 18 Amino Acid Oxidation and the Production of Urea670 * Required to some degree in young, growing animals, and/or sometimes during illness. Conditionally Nonessential essential* Essential Alanine Arginine Histidine Asparagine Cysteine Isoleucine Aspartate Glutamine Leucine Glutamate Glycine Lysine Serine Proline Methionine Tyrosine Phenylalanine Threonine Tryptophan Valine TABLE 18–1 Nonessential and Essential Amino Acids for Humans and the Albino Rat C O CoA - SH S - CoA Benzoyl-CoA Benzoate COO H11002 ATP AMP H11001 PP i Glycine CoA - SH C O Hippurate (benzoylglycine) H11001 CoA - SH CoA - SH Acetyl - CoA H11001 Phenylacetate Phenylbutyrate CH 2 CH 2 COO H11002 CH 2 CH 2 CH 2 COO H11002 NH CH 2 COO H11002 CH 2 COO H11002 Glutamine CoA - SH C O S - CoA Phenylacetyl-CoA Phenylacetylglutamine ATP AMP H11001 PP i CH 2 CH 2 NH 2 CH 2 CHC O C O COO H11002 NH H 3 N H11001 H 3 N H11001 CH 2 NH 2 CH 2 CH C O COO H11002 H9252 oxidation FIGURE 18–14 Treatment for deficiencies in urea cycle en- zymes. The aromatic acids benzoate and phenylbutyrate, ad- ministered in the diet, are metabolized and combine with glycine and glutamine, respectively. The products are excreted in the urine. Sub- sequent synthesis of glycine and glutamine to replenish the pool of these intermediates removes ammonia from the bloodstream. 8885d_c18_670 2/3/04 4:13 PM Page 670 mac76 mac76:385_reb: SUMMARY 18.2 Nitrogen Excretion and the Urea Cycle ■ Ammonia is highly toxic to animal tissues. In the urea cycle, ornithine combines with ammonia, in the form of carbamoyl phosphate, to form citrulline. A second amino group is transferred to citrulline from aspartate to form arginine—the immediate precursor of urea. Arginase catalyzes hydrolysis of arginine to urea and ornithine; thus ornithine is regenerated in each turn of the cycle. ■ The urea cycle results in a net conversion of oxaloacetate to fumarate, both of which are intermediates in the citric acid cycle. The two cycles are thus interconnected. ■ The activity of the urea cycle is regulated at the level of enzyme synthesis and by allosteric regulation of the enzyme that catalyzes the formation of carbamoyl phosphate. 18.3 Pathways of Amino Acid Degradation The pathways of amino acid catabolism, taken together, normally account for only 10% to 15% of the human body’s energy production; these pathways are not nearly as active as glycolysis and fatty acid oxidation. Flux through these catabolic routes also varies greatly, de- pending on the balance between requirements for bio- synthetic processes and the availability of a particular amino acid. The 20 catabolic pathways converge to form only six major products, all of which enter the citric acid cycle (Fig. 18–15). From here the carbon skeletons are diverted to gluconeogenesis or ketogenesis or are com- pletely oxidized to CO 2 and H 2 O. All or part of the carbon skeletons of seven amino acids are ultimately broken down to acetyl-CoA. Five amino acids are converted to H9251-ketoglutarate, four to succinyl-CoA, two to fumarate, and two to oxaloacetate. Parts or all of six amino acids are converted to pyru- vate, which can be converted to either acetyl-CoA or oxaloacetate. We later summarize the individual path- ways for the 20 amino acids in flow diagrams, each lead- ing to a specific point of entry into the citric acid cycle. In these diagrams the carbon atoms that enter the cit- ric acid cycle are shown in color. Note that some amino acids appear more than once, reflecting different fates for different parts of their carbon skeletons. Rather than examining every step of every pathway in amino acid catabolism, we single out for special discussion some en- zymatic reactions that are particularly noteworthy for their mechanisms or their medical significance. Some Amino Acids Are Converted to Glucose, Others to Ketone Bodies The seven amino acids that are degraded entirely or in part to acetoacetyl-CoA and/or acetyl-CoA—phenylala- nine, tyrosine, isoleucine, leucine, tryptophan, threo- nine, and lysine—can yield ketone bodies in the liver, 18.3 Pathways of Amino Acid Degradation 671 Glucose Fumarate Succinyl-CoA Citrate CO 2 Isocitrate Succinate Citric acid cycle -Ketoglutarate Phenylalanine Tyrosine Glutamate Arginine Glutamine Histidine Proline Isoleucine Methionine Threonine Valine Ketone bodies Oxaloacetate Malate Glucogenic Ketogenic Acetyl-CoA Pyruvate Alanine Cysteine Glycine Serine Threonine Tryptophan Acetoacetyl-CoA Leucine Lysine Phenylalanine Tryptophan Tyrosine Asparagine Aspartate Isoleucine Leucine Threonine Tryptophan H9251 FIGURE 18–15 Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded via at least two different pathways (see Figs 18–19, 18–27), and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic. 8885d_c18_656-689 2/3/04 11:39 AM Page 671 mac76 mac76:385_reb: where acetoacetyl-CoA is converted to acetoacetate and then to acetone and H9252-hydroxybutyrate (see Fig. 17–18). These are the ketogenic amino acids (Fig. 18–15). Their ability to form ketone bodies is particularly evi- dent in uncontrolled diabetes mellitus, in which the liver produces large amounts of ketone bodies from both fatty acids and the ketogenic amino acids. The amino acids that are degraded to pyruvate, H9251- ketoglutarate, succinyl-CoA, fumarate, and/or oxaloac- etate can be converted to glucose and glycogen by path- ways described in Chapters 14 and 15. They are the glucogenic amino acids. The division between keto- genic and glucogenic amino acids is not sharp; five amino acids—tryptophan, phenylalanine, tyrosine, thre- onine, and isoleucine—are both ketogenic and gluco- genic. Catabolism of amino acids is particularly critical to the survival of animals with high-protein diets or dur- ing starvation. Leucine is an exclusively ketogenic amino acid that is very common in proteins. Its degradation makes a substantial contribution to ketosis under star- vation conditions. Several Enzyme Cofactors Play Important Roles in Amino Acid Catabolism A variety of interesting chemical rearrangements occur in the catabolic pathways of amino acids. It is useful to begin our study of these pathways by noting the classes of reactions that recur and introducing their enzyme co- factors. We have already considered one important class: transamination reactions requiring pyridoxal phosphate. Another common type of reaction in amino acid catabolism is one-carbon transfers, which usually involve one of three cofactors: biotin, tetrahydrofolate, or S-adenosylmethionine (Fig. 18–16). These cofactors transfer one-carbon groups in different oxidation states: biotin transfers carbon in its most oxidized state, CO 2 (see Fig. 14–18); tetrahydrofolate transfers one-carbon groups in intermediate oxidation states and sometimes as methyl groups; and S-adenosylmethionine transfers methyl groups, the most reduced state of carbon. The latter two cofactors are especially important in amino acid and nucleotide metabolism. Tetrahydrofolate (H 4 folate), synthesized in bac- teria, consists of substituted pterin (6-methylpterin), p-aminobenzoate, and glutamate moieties (Fig. 18–16). The oxidized form, folate, is a vitamin for mammals; it is converted in two steps to tetrahydrofolate by the en- zyme dihydrofolate reductase. The one-carbon group undergoing transfer, in any of three oxidation states, is bonded to N-5 or N-10 or both. The most reduced form of the cofactor carries a methyl group, a more oxidized form carries a methylene group, and the most oxidized forms carry a methenyl, formyl, or formimino group (Fig. 18–17). Most forms of tetrahydrofolate are inter- convertible and serve as donors of one-carbon units in a variety of metabolic reactions. The primary source of one-carbon units for tetrahydrofolate is the carbon re- moved in the conversion of serine to glycine, producing N 5 , N 10 -methylenetetrahydrofolate. Although tetrahydrofolate can carry a methyl group at N-5, the transfer potential of this methyl group is in- sufficient for most biosynthetic reactions. S-Adenosyl- methionine (adoMet) is the preferred cofactor for bi- ological methyl group transfers. It is synthesized from ATP and methionine by the action of methionine H 2 NN O NH Pterin N RN Chapter 18 Amino Acid Oxidation and the Production of Urea672 S Biotin COO H11002 O HN HN CH NH HC C CH 2 CH H 2 C H 2 CH 2 CH 2 9 7 4a 5 1 3 4 6 8 CH 2 N O CH N N N H 2 H C H H H HCH 2 COO H11002 NH CH 2 COO H11002 p-aminobenzoate N CH 2 O CH 2 H11001 HC CH 2 H 3 N COO H11002 H11001 S-Adenosylmethionine (adoMet) methionine OH N N H N H H O OH H NH S 2 CH 3 N CH 2 adenosine Tetrahydrofolate (H 4 folate) valerate glutamate 10 8a 6-methylpterin FIGURE 18–16 Some enzyme cofactors important in one-carbon transfer reactions. The nitrogen atoms to which one-carbon groups are attached in tetrahydrofolate are shown in blue. 8885d_c18_656-689 2/3/04 11:39 AM Page 672 mac76 mac76:385_reb: adenosyl transferase (Fig. 18–18, step 1 ). This re- action is unusual in that the nucleophilic sulfur atom of methionine attacks the 5H11032 carbon of the ribose moiety of ATP rather than one of the phosphorus atoms. Tri- phosphate is released and is cleaved to P i and PP i on the enzyme, and the PP i is cleaved by inorganic pyro- phosphatase; thus three bonds, including two bonds of high-energy phosphate groups, are broken in this reac- tion. The only other known reaction in which triphos- phate is displaced from ATP occurs in the synthesis of coenzyme B 12 (see Box 17–2, Fig. 3). S-Adenosylmethionine is a potent alkylating agent by virtue of its destabilizing sulfonium ion. The methyl group is subject to attack by nucleophiles and is about 18.3 Pathways of Amino Acid Degradation 673 H N N H N H CH 2 CH 2 5 10 N 10 -formyl- tetrahydrofolate synthetase ADP H11001 P i ADP H11001 P i H N CH 3 H N H CH 2 CH 2 5 10 Oxidation state (group transferred) COO H11002 NADH H11001 H H11001 H N H 2 C N H N CH 2 CH 2 5 10 N 5 ,N 10 -methylene- tetrahydrofolate reductase NADP H11001 NADPH H11001 H H11001 H N N H N H CH 2 5 10 N 5 -Formimino- tetrahydrofolate HC C O H CH 2 CH 2 OH H N N H N H CH 2 CH 2 5 10 O H N 5 -Formyl- tetrahydrofolate H N N H N CH 2 5 10 N 5 ,N 10 -methylene- tetrahydrofolate dehydrogenase N 5 ,N 10 -methenyl- tetrahydrofolate synthetase cyclohydrolase (minor); spontaneous HC CH 2 HN O CH (most oxidized) (most reduced) CH 2 OH NAD H11001 NH 4 H 2 O CHH 3 N H H11001 COO H11002 CHH 3 N H11001 GlycineSerine PLP H 2 O H N N H N H CH 2 CH 2 5 10 N 5 -Methyl- tetrahydrofolate CH 3 N H serine hydroxymethyl transferase cyclodeaminase Tetrahydrofolate H11001 H11001 N 5 ,N 10 -Methylene- tetrahydrofolate N 10 -Formyl- tetrahydrofolate N 5 ,N 10 -Methenyl- tetrahydrofolate Formate cyclohydrolase ATP ATP FIGURE 18–17 Conversions of one-carbon units on tetrahydrofolate. The different molecular species are grouped according to oxidation state, with the most reduced at the top and most oxidized at the bot- tom. All species within a single shaded box are at the same oxidation state. The conversion of N 5 ,N 10 -methylenetetrahydrofolate to N 5 - methyltetrahydrofolate is effectively irreversible. The enzymatic trans- fer of formyl groups, as in purine synthesis (see Fig. 22–33) and in the formation of formylmethionine in prokaryotes (Chapter 27), generally uses N 10 -formyltetrahydrofolate rather than N 5 -formyltetrahydrofolate. The latter species is significantly more stable and therefore a weaker donor of formyl groups. N 5 -formyltetrahydrofolate is a minor byprod- uct of the cyclohydrolase reaction, and can also form spontancously. Conversion of N 5 -formyltetrahydrofolate to N 5 , N 10 -methenyltetrahy- drofolate, requires ATP, because of an otherwise unfavorable equilib- rium. Note that N 5 -formiminotetrahydrofolate is derived from histidine in a pathway shown in Figure 18–26. 8885d_c18_656-689 2/3/04 11:39 AM Page 673 mac76 mac76:385_reb: 1,000 times more reactive than the methyl group of N 5 - methyltetrahydrofolate. Transfer of the methyl group from S-adenosylmethi- onine to an acceptor yields S-adenosylhomocysteine (Fig. 18–18, step 2 ), which is subsequently broken down to homocysteine and adenosine (step 3 ). Methionine is regenerated by transfer of a methyl group to homo- cysteine in a reaction catalyzed by methionine synthase (step 4 ), and methionine is reconverted to S-adenosyl- methionine to complete an activated-methyl cycle. One form of methionine synthase common in bacteria uses N 5 -methyltetrahydrofolate as a methyl donor. Another form of the enzyme present in some bacteria and mammals uses N 5 -methyltetrahydro- folate, but the methyl group is first transferred to cobal- amin, derived from coenzyme B 12 , to form methyl- cobalamin as the methyl donor in methionine formation. This reaction and the rearrangement of L-methyl- malonyl-CoA to succinyl-CoA (see Box 17–2, Fig. 1a) are the only known coenzyme B 12 –dependent reactions in mammals. In cases of vitamin B 12 deficiency, some symptoms can be alleviated by administering not only vitamin B 12 but folate. As noted above, the methyl group of methylcobalamin is derived from N 5 -methyltetrahy- drofolate. Because the reaction converting the N 5 ,N 10 - methylene form to the N 5 -methyl form of tetrahydrofo- late is irreversible (Fig. 18–17), if coenzyme B 12 is not available for the synthesis of methylcobalamin, then no acceptor is available for the methyl group of N 5 -methyl- tetrahydrofolate and metabolic folates become trapped in the N 5 -methyl form. This sequestering of folates in one form may be the cause of some symptoms of the vi- tamin B 12 deficiency disease pernicious anemia. How- ever, we do not know whether this is the only effect of insufficient vitamin B 12 . ■ Tetrahydrobiopterin, another cofactor of amino acid catabolism, is similar to the pterin moiety of tetrahydrofolate, but it is not involved in one-carbon transfers; instead it participates in oxidation reactions. We consider its mode of action when we discuss phenyl- alanine degradation (see Fig. 18–24). Six Amino Acids Are Degraded to Pyruvate The carbon skeletons of six amino acids are converted in whole or in part to pyruvate. The pyruvate can then be converted to either acetyl-CoA (a ketone body precur- sor) or oxaloacetate (a precursor for gluconeogenesis). Thus amino acids catabolized to pyruvate are both ke- togenic and glucogenic. The six are alanine, tryptophan, cysteine, serine, glycine, and threonine (Fig. 18–19). Alanine yields pyruvate directly on transamination with Chapter 18 Amino Acid Oxidation and the Production of Urea674 H11002 OCH 2 P O O H11002 O H11002 O H11002 OO OP POO Methionine H 2 O H H N H N CH 3 CH 2 5 N 5 -Methyltetrahydrofolate N H H N H N H CH 2 CH 2 N H CH 2 Tetrahydrofolate ATP CH 3 N S CH 2 COO H11002 CH 2 CH 3 NH 2 OH N O H HH H OH N N N methionine synthase Adenosine coenzyme B 12 CH 3 N H11001 SH CH 2 H COO H11002 CH 2 Homocysteine hydrolase CH 3 N H11001 Adenosine H COO H11002 CH 2 S-Adenosyl- homocysteine S CH 2 H11001 H11001 NH 2 CH 3 OH N O H H H H OH N N N CH 3 N H11001 H11001 S CH 2 COO H11002 CH 2 H CH 3 S-Adenosyl- methionine CH 2 a variety of methyl transferases R R 2 PP i H11001 P i methionine adenosyl transferase 1 3 4 FIGURE 18–18 Synthesis of methionine and S-adenosylmethionine in an activated-methyl cycle. The steps are described in the text. In the methionine synthase reaction (step 4 ), the methyl group is trans- ferred to cobalamin to form methylcobalamin, which in turn is the methyl donor in the formation of methionine. S-Adenosylmethionine, which has a positively charged sulfur (and is thus a sulfonium ion), is a powerful methylating agent in a number of biosynthetic reactions. The methyl group acceptor (step 2 ) is designated R. 8885d_c18_656-689 2/3/04 11:39 AM Page 674 mac76 mac76:385_reb: H9251-ketoglutarate, and the side chain of tryptophan is cleaved to yield alanine and thus pyruvate. Cysteine is converted to pyruvate in two steps; one removes the sulfur atom, the other is a transamination. Serine is converted to pyruvate by serine dehydratase. Both the H9252-hydroxyl and the H9251-amino groups of serine are re- moved in this single pyridoxal phosphate–dependent re- action (Fig. 18–20a). Glycine is degraded via three pathways, only one of which leads to pyruvate. Glycine is converted to ser- ine by enzymatic addition of a hydroxymethyl group (Figs 18–19 and 18–20b). This reaction, catalyzed by serine hydroxymethyl transferase, requires the coenzymes tetrahydrofolate and pyridoxal phosphate. The serine is converted to pyruvate as described above. In the second pathway, which predominates in animals, glycine undergoes oxidative cleavage to CO 2 , NH 4 H11001 , and a methylene group (OCH 2 O) (Fig. 18–19). This read- ily reversible reaction, catalyzed by glycine cleavage enzyme (also called glycine synthase), also requires tetrahydrofolate, which accepts the methylene group. In this oxidative cleavage pathway the two carbon atoms of glycine do not enter the citric acid cycle. One carbon is lost as CO 2 and the other becomes the methylene group of N 5 ,N 10 -methylenetetrahydrofolate (Fig. 18–17), a one- carbon group donor in certain biosynthetic pathways. This second pathway for glycine degradation ap- pears to be critical in mammals. Humans with se- rious defects in glycine cleavage enzyme activity suffer from a condition known as nonketotic hyperglycinemia. The condition is characterized by elevated serum levels of glycine, leading to severe mental deficiencies and death in very early childhood. At high levels, glycine is an inhibitory neurotransmitter, perhaps explaining the neurological effects of the disease. Many genetic defects of amino acid metabolism have been identified in hu- mans (Table 18–2). We will encounter several more in this chapter. ■ In the third and final pathway of glycine degrada- tion, the achiral glycine molecule is a substrate for the enzyme D-amino acid oxidase. The glycine is converted to glyoxylate, an alternative substrate for hepatic lactate 18.3 Pathways of Amino Acid Degradation 675 CH 3 CH 2 CH 3 CO 2 NH 4 H11001 CH OH CH COO H11002 COO H11002 NH 3 H11001 NAD H11001 NADH NAD H11001 NADH CoA N 5 , N 10 -methylene H 4 folate 2-Amino-3-ketobutyrate threonine dehydrogenase 2-amino- 3-ketobutyrate CoA ligase serine hydroxy- methyl transferase serine dehydratase alanine aminotransferase glycine cleavage enzyme NH 3 H11001 NH 3 H11001 CH COO H11002 Threonine CH 2 H 2 O H 2 O CH COO H11002 NH 3 H11001 NH 4 H11001 HO Serine Cysteine Glycine PLP PLP Glutamate 2 steps C O CH 3 COO H11002 C O CH 2 CH SH COO H11002 NH 3 H11001 Acetyl-CoA Pyruvate H11001 H 4 folate H9251-Ketoglutarate H CH 3 CH 2 COO H11002 CH CH COO H11002 N Tryptophan Alanine NH 3 NH 3 H11001 H11001 4 steps PLP FIGURE 18–19 Catabolic pathways for alanine, glycine, serine, cysteine, tryptophan, and threonine. The fate of the indole group of tryptophan is shown in Figure 18–21. Details of most of the reactions involving serine and glycine are shown in Figure 18–20. The pathway for threonine degradation shown here accounts for only about a third of threonine catabolism (for the alternative pathway, see Fig. 18–27). Several pathways for cysteine degradation lead to pyruvate. The sulfur of cysteine has several alternative fates, one of which is shown in Figure 22–15. Carbon atoms here and in subsequent figures are color-coded as necessary to trace their fates. 8885d_c18_656-689 2/3/04 11:39 AM Page 675 mac76 mac76:385_reb: Chapter 18 Amino Acid Oxidation and the Production of Urea676 Covalently bound serine Pyruvate (b) Serine hydroxymethyltransferase reaction 1 H 2 O H 2 O NH 3 2 Enz Enz P Enz H Enz NH H + CH + : : COO – OH CH 2 CH BPLP NHCH + COO – CH 2 CHB Lys PLP PLP HB Enz Enz Lys PLP H 2 N + COO – CH 3 C O COO – CH 2 C H 2 N COO – H + CH 2 C 2 3 Enz Enz Lys PLP 4 5 N 5 , N 10 -methylene H 4 folate H 4 folate NADH NAD + 4 3 Covalently bound glycine PLP-stabilized carbanion PLP-stabilized carbanion Serine 1 NHCH + : COO – H CH BPLP H 3 NH + COO – CH 2 OH C H COO – CH 2 HO N 5 , N 10 -methylene H 4 folate C NHCH + – COO – HCPLP NHCH + PLP 3 2 H 4 folate (c) Glycine cleavage enzyme reaction Covalently bound glycine 1 NH HCH + C OO – H C CO 2 PLP NHCH + C H H C H H H + PLP S S (a) Serine dehydratase reaction Enz H NHCH + PLP S HS Enz H Enz T Enz L H 2 NCH 2 NH 3 S HS Enz H HS HS Enz H S S – MECHANISM FIGURE 18–20 Interplay of the pyridoxal phosphate and tetrahydrofolate cofactors in serine and glycine metabolism. The first step in each of these reactions (not shown) involves the forma- tion of a covalent imine linkage between enzyme-bound PLP and the substrate amino acid—serine in (a), glycine in (b) and (c). (a) The ser- ine dehydratase reaction entails a PLP-catalyzed elimination of water across the bond between the H9251 and H9252 carbons (step 1 ), leading even- tually to the production of pyruvate (steps 2 through 4 ). (b) In the serine hydroxymethyltransferase reaction, a PLP-stabilized carbanion on the H9251 carbon of glycine (product of step 1 ) is a key intermediate in the transfer of the methylene group (as OCH 2 OOH) from N 5 ,N 10 - methylenetetrahydrofolate to form serine. This reaction is reversible. (c) The glycine cleavage enzyme is a multienzyme complex, with com- ponents P, H, T, and L. The overall reaction, which is reversible, con- verts glycine to CO 2 and NH 4 H11001 , with the second glycine carbon taken up by tetrahydrofolate to form N 5 ,N 10 -methylenetetrahydrofolate. Pyri- doxal phosphate activates the H9251 carbon of amino acids at critical stages in all these reactions, and tetrahydrofolate carries one-carbon units in two of them (see Figs 18–6, 18–17). 8885d_c18_656-689 2/3/04 11:39 AM Page 676 mac76 mac76:385_reb: dehydrogenase (p. 538). Glyoxylate is oxidized in an NAD H11001 -dependent reaction to oxalate: The primary function of D-amino acid oxidase, present at high levels in the kidney, is thought to be the detoxification of ingested D-amino acids derived from bacterial cell walls and from cooked foodstuffs (heat causes some spontaneous racemization of the L- amino acids in proteins). Oxalate, whether obtained in foods or produced enzymatically in the kidneys, has medical significance. Crystals of calcium oxalate ac- count for up to 75% of all kidney stones. ■ There are two significant pathways for threonine degradation. One pathway leads to pyruvate via glycine (Fig. 18–19). The conversion to glycine occurs in two steps, with threonine first converted to 2-amino-3- ketobutyrate by the action of threonine dehydrogenase. This is a relatively minor pathway in humans, account- ing for 10% to 30% of threonine catabolism, but is more important in some other mammals. The major pathway in humans leads to succinyl-CoA and is described later. In the laboratory, serine hydroxymethyltransferase will catalyze the conversion of threonine to glycine and acetaldehyde in one step, but this is not a significant pathway for threonine degradation in mammals. Seven Amino Acids Are Degraded to Acetyl-CoA Portions of the carbon skeletons of seven amino acids— tryptophan, lysine, phenylalanine, tyrosine, leucine, isoleucine, and threonine—yield acetyl-CoA and/or acetoacetyl-CoA, the latter being converted to acetyl- CoA (Fig. 18–21). Some of the final steps in the degrada- tive pathways for leucine, lysine, and tryptophan re- semble steps in the oxidation of fatty acids. Threonine (not shown in Fig. 18–21) yields some acetyl-CoA via the minor pathway illustrated in Figure 18–19. The degradative pathways of two of these seven amino acids deserve special mention. Tryptophan break- down is the most complex of all the pathways of amino 18.3 Pathways of Amino Acid Degradation 677 TABLE 18–2 Some Human Genetic Disorders Affecting Amino Acid Catabolism Approximate incidence (per 100,000 Medical condition births) Defective process Defective enzyme Symptoms and effects Albinism H110213 Melanin synthesis Tyrosine 3- Lack of pigmentation: from tyrosine monooxygenase white hair, pink skin (tyrosinase) Alkaptonuria H110210.4 Tyrosine degradation Homogentisate Dark pigment in urine; 1,2-dioxygenase late-developing arthritis Argininemia H110210.5 Urea synthesis Arginase Mental retardation Argininosuccinic H110211.5 Urea synthesis Argininosuccinase Vomiting; convulsions acidemia Carbamoyl phosphate H110210.5 Urea synthesis Carbamoyl phosphate Lethargy; convulsions; synthetase I synthetase I early death deficiency Homocystinuria H110210.5 Methionine degradation Cystathionine H9252-synthase Faulty bone develop- ment; mental retardation Maple syrup urine H110210.4 Isoleucine, leucine, and Branched-chain H9251-keto Vomiting; convulsions; disease (branched- valine degradation acid dehydrogenase mental retardation; chain ketoaciduria) complex early death Methylmalonic H110210.5 Conversion of propionyl- Methylmalonyl-CoA Vomiting; convulsions; acidemia CoA to succinyl-CoA mutase mental retardation; early death Phenylketonuria H110218 Conversion of phenyl- Phenylalanine hydroxylase Neonatal vomiting; alanine to tyrosine mental retardation H11001 ONH 3 NH 3 CH 2 COO H11002 COO H11002 O 2 H 2 O CH NAD H11001 NADH Glycine Glyoxylate Oxalate D-amino acid oxidase COO H11002 COO H11002 8885d_c18_677 2/3/04 4:14 PM Page 677 mac76 mac76:385_reb: acid catabolism in animal tissues; portions of tryptophan (four of its carbons) yield acetyl-CoA via acetoacetyl- CoA. Some of the intermediates in tryptophan catabolism are precursors for the synthesis of other biomolecules (Fig. 18–22), including nicotinate, a precursor of NAD and NADP in animals; serotonin, a neurotransmitter in vertebrates; and indoleacetate, a growth factor in plants. Some of these biosynthetic pathways are described in more detail in Chapter 22 (see Figs 22–28, 22–29). The breakdown of phenylalanine is noteworthy be- cause genetic defects in the enzymes of this pathway lead to several inheritable human diseases (Fig. 18–23), as discussed below. Phenylalanine and its oxidation product tyrosine (both with nine carbons) are degraded into two fragments, both of which can enter the citric acid cycle: four of the nine carbon atoms yield free ace- toacetate, which is converted to acetoacetyl-CoA and thus acetyl-CoA, and a second four-carbon fragment is recovered as fumarate. Eight of the nine carbons of these two amino acids thus enter the citric acid cycle; the remaining carbon is lost as CO 2 . Phenylalanine, af- ter its hydroxylation to tyrosine, is also the precursor of dopamine, a neurotransmitter, and of norepinephrine and epinephrine, hormones secreted by the adrenal medulla (see Fig. 22–29). Melanin, the black pigment of skin and hair, is also derived from tyrosine. FIGURE 18–21 Catabolic pathways for tryptophan, lysine, phenyl- alanine, tyrosine, leucine, and isoleucine. These amino acids donate some of their carbons (red) to acetyl-CoA. Tryptophan, phenylalanine, tyrosine, and isoleucine also contribute carbons (blue) to pyruvate or citric acid cycle intermediates. The phenylalanine pathway is de- scribed in more detail in Figure 18–23. The fate of nitrogen atoms is not traced in this scheme; in most cases they are transferred to H9251- ketoglutarate to form glutamate. C 9 steps N H Pyruvate CH 3 C C C C CH CH 3 C C O Leucine C C COO H11002 H11001 NH 3 CH 3 CH COO H11002 H11001 2CO 2 COO H11002 Alanine Tryptophan CH 2 CH COO H11002 H11001 NH 3 CH 2 C CC HO C Phenylalanine C CH COO H11002 H11001 NH 3 CH 2 C CC C C O CO 2 CO 2 CH 3 CH 3 Tyrosine CH COO H11002 H11001 NH 3 CH COO H11002 Lysine CH 2 H11001 H 3 N H11001 CH 2 CH 2 CO 2 NH 3 CHCH COO H11002 H9251-Ketoadipate C Isoleucine CH 2 H11002 OOC CH 2 CH 2 CoA-SH CoA-SH CoA-SH H11002 OOC CH 2 Glutaryl-CoA O CH 2 NH 3 H11001 NAD C COO H11002 CH 2 O CH 3 CoA-SH C CoACH 2 H11002 OOC CH 2 CH 2 S- C CoA CH 3 CH 2 O S-C O CH 3 CH 2 CH COO H11002 CH NH 3 H11001 COO H11002 O CH 3 CH 2 CH 3 CH S- CoA Acetoacetyl-CoA CoA Propionyl-CoA Acetoacetate 3 steps NADH CO 2 CO 2 O 5 steps 4 steps 4 steps 6 steps S- Fumarate Acetyl-CoA Succinyl-CoA Chapter 18 Amino Acid Oxidation and the Production of Urea678 8885d_c18_656-689 2/3/04 11:39 AM Page 678 mac76 mac76:385_reb: Phenylalanine Catabolism Is Genetically Defective in Some People Given that many amino acids are either neuro- transmitters or precursors or antagonists of neutrotransmitters, genetic defects of amino acid me- tabolism can cause defective neural development and mental retardation. In most such diseases specific inter- mediates accumulate. For example, a genetic defect in phenylalanine hydroxylase, the first enzyme in the catabolic pathway for phenylalanine (Fig. 18–23), is re- sponsible for the disease phenylketonuria (PKU), the most common cause of elevated levels of phenylalanine (hyperphenylalaninemia). Phenylalanine hydroxylase (also called phenylala- nine-4-monooxygenase) is one of a general class of en- zymes called mixed-function oxidases (see Box 21–1), all of which catalyze simultaneous hydroxylation of a substrate by an oxygen atom of O 2 and reduction of the other oxygen atom to H 2 O. Phenylalanine hydroxylase 679 H 2 O H H11001 COO H11002 H11001 CH 2 O 2 3-ketoacyl-CoA transferase H11002 OOC H11001 NH 3 CH C COO H11002 CH 2 C Phenylalanine NAD H11001 NADH H11001 H H11001 tetrahydrobiopterin O 2 phenylalanine hydroxylase Tyrosinemia III PKU H11001 NH 3 CH C COO H11002 CH 2 HO C Tyrosine Glutamate tyrosine aminotransferase -Ketoglutarate Tyrosinemia II C C COO H11002 CH 2 p-Hydroxyphenylpyruvate HO O C CCOO H11002 CH 2 Homogentisate OH C HO CO 2 p-hydroxyphenylpyruvate dioxygenase O 2 Fumarate OH H C CH 2 C CH 3 COO H11002 C O Alkaptonuria C homogentisate 1,2-dioxygenase C H Homogentisate C COO H11002 CH 2 maleylacetoacetate isomerase O Maleylacetoacetate H CC CH 2 O C H C COO H11002 CH 2 fumarylacetoacetase O Fumarylacetoacetate H CC CH 2 O C H Acetoacetyl-CoA CH 2 CCH 3 O COO H11002 C CoA O S- Acetoacetate HO H11002 OOC H11002 OOC Tyrosinemia I Succinyl-CoA Succinate H 2 O FIGURE 18–23 Catabolic pathways for phenylalanine and tyrosine. In humans these amino acids are normally con- verted to acetoacetyl-CoA and fumarate. Genetic defects in many of these enzymes cause inheritable human diseases (shaded yellow). N H C HC HC COO H11002 Nicotinate (niacin), a precursor of NAD and NADP C CH COO H11002 CH 2 H N Indoleacetate, a plant growth factor C C H H C HC HC CH C C N H Tryptophan CH 2 COO H11002 NH 3 H11001 H NH 3 CH 2 H11001 N Serotonin, a neurotransmitter HO CH 2 FIGURE 18–22 Tryptophan as precursor. The aromatic rings of tryp- tophan give rise to nicotinate, indoleacetate, and serotonin. Colored atoms trace the source of the ring atoms in nicotinate. 8885d_c18_656-689 2/3/04 11:39 AM Page 679 mac76 mac76:385_reb: requires the cofactor tetrahydrobiopterin, which carries electrons from NADH to O 2 and becomes oxidized to dihydrobiopterin in the process (Fig. 18–24). It is sub- sequently reduced by the enzyme dihydrobiopterin reductase in a reaction that requires NADH. In individuals with PKU, a secondary, normally little-used pathway of phenylalanine metabolism comes into play. In this pathway phenylalanine undergoes transamination with pyruvate to yield phenylpyruvate (Fig. 18–25). Phenylalanine and phenylpyruvate accu- mulate in the blood and tissues and are excreted in the urine—hence the name “phenylketonuria.” Much of the phenylpyruvate, rather than being excreted as such, is either decarboxylated to phenylacetate or reduced to phenyllactate. Phenylacetate imparts a characteristic odor to the urine, which nurses have traditionally used to detect PKU in infants. The accumulation of phenyl- alanine or its metabolites in early life impairs normal development of the brain, causing severe mental retar- dation. This may be caused by excess phenylalanine competing with other amino acids for transport across the blood-brain barrier, resulting in a deficit of required metabolites. Phenylketonuria was among the first inheritable metabolic defects discovered in humans. When this con- dition is recognized early in infancy, mental retardation can largely be prevented by rigid dietary control. The diet must supply only enough phenylalanine and tyro- sine to meet the needs for protein synthesis. Consump- tion of protein-rich foods must be curtailed. Natural pro- teins, such as casein of milk, must first be hydrolyzed and much of the phenylalanine removed to provide an appropriate diet, at least through childhood. Because the artificial sweetener aspartame is a dipeptide of aspartate and the methyl ester of phenylalanine (see Fig. 1–23b), foods sweetened with aspartame bear warnings ad- dressed to individuals on phenylalanine-controlled diets. Phenylketonuria can also be caused by a defect in the enzyme that catalyzes the regeneration of tetrahy- drobiopterin (Fig. 18–24). The treatment in this case is more complex than restricting the intake of phenylala- nine and tyrosine. Tetrahydrobiopterin is also required for the formation of L-3,4-dihydroxyphenylalanine (L- dopa) and 5-hydroxytryptophan—precursors of the neurotransmitters norepinephrine and serotonin, respec- tively—and in phenylketonuria of this type, these pre- cursors must be supplied in the diet. Supplementing the diet with tetrahydrobiopterin itself is ineffective because it is unstable and does not cross the blood-brain barrier. Chapter 18 Amino Acid Oxidation and the Production of Urea680 N H O CH 3 N H HNH CH OH CH OH H 2 N N H H 7,8-Dihydrobiopterin (quinoid form) N H O N H NH CH HO CH OH N H H Tyrosine NH H H CH 3 CHCH 2 COO H11002 NH 3 H11001 CH O 2 COO H11002 NH 3 H11001 OH 5,6,7,8-Tetrahydrobiopterin Phenylalanine dihydrobiopterin reductase CH 2 H 2 O phenylalanine hydroxylase NADH H11001 H H11001 NAD H11001 8 6 5 7 FIGURE 18–24 Role of tetrahydrobiopterin in the phenylalanine hy- droxylase reaction. The H atom shaded pink is transferred directly from C-4 to C-3 in the reaction. This feature, discovered at the NIH, is called the NIH Shift. CH COO H11002 C O H11001 Phenylalanine Pyruvate Alanine Phenylpyruvate Phenylacetate Phenyllactate PLP CH 2 NH 3 OH CH 3 CH 2 CH 2 CH 2 COO H11002 C COO H11002 COO H11002 CH COO H11002 O COO H11002 CH 3 CH aminotransferase H11001 NH 3 CO 2 H 2 O FIGURE 18–25 Alternative pathways for catabolism of phenylalanine in phenylketonuria. In PKU, phenylpyruvate accumulates in the tissues, blood, and urine. The urine may also con- tain phenylacetate and phenyllactate. 8885d_c18_656-689 2/3/04 11:39 AM Page 680 mac76 mac76:385_reb: Screening newborns for genetic diseases can be highly cost-effective, especially in the case of PKU. The tests (no longer relying on urine odor) are relatively in- expensive, and the detection and early treatment of PKU in infants (eight to ten cases per 100,000 new- borns) saves millions of dollars in later health care costs each year. More importantly, the emotional trauma avoided by early detection with these simple tests is inestimable. Another inheritable disease of phenylalanine catab- olism is alkaptonuria, in which the defective enzyme is homogentisate dioxygenase (Fig. 18–23). Less se- rious than PKU, this condition produces few ill effects, although large amounts of homogentisate are excreted and its oxidation turns the urine black. Individuals with alkaptonuria are also prone to develop a form of arthri- tis. Alkaptonuria is of considerable historical interest. Archibald Garrod discovered in the early 1900s that this condition is inherited, and he traced the cause to the absence of a single enzyme. Garrod was the first to make a connection between an inheritable trait and an en- zyme, a great advance on the path that ultimately led to our current understanding of genes and the infor- mation pathways described in Part III. ■ Five Amino Acids Are Converted to H9251-Ketoglutarate The carbon skeletons of five amino acids (proline, glu- tamate, glutamine, arginine, and histidine) enter the cit- ric acid cycle as H9251-ketoglutarate (Fig. 18–26). Proline, glutamate, and glutamine have five-carbon skeletons. The cyclic structure of proline is opened by oxidation 18.3 Pathways of Amino Acid Degradation 681 NH COO H11002 H 3 N H11001 H C C H 2 C C NH Glutamate -semialdehydeH9253 COO H11002 H 3 N H11001 H 2 NH 3 H11001 C H H 2 C H 2 C C Glutamine ornithine H9254-aminotransferase COO H11002 H 3 N H11001 H 2 H 3 H11001 C H H 2 O H 2 O C H 2 C C Ornithine (uncatalyzed) H 2 N 5 -Formimino O H COO H11002 C H CH 2 C H 2 H 2 N H11001 H 2 COO H11002 CHC CC H 2 H 2 H H 2 O H 2 glutaminase Proline NADPH H11001 H H11001 glutamate dehydrogenase H9004 1 -Pyrroline- 5-carboxylate O2 1 2 H 2 O proline oxidase NADP H11001 NH 3 H11001 CH COO H11002 H 2 C C Arginine H 3 N H 2 2 C C H COO H11002 CH 2 H11001 COO H11002 Histidine H 2 O 3 H 4 folate 14 H 2 O 2 H9251-Keto- glutarate Glutamate NH 4 H 4 folate OC H H 3 N H 2 C C H COO H11002 CH 2 H11001 N H9251-Ketoglutarate Urea arginase H 2 O H11001 NH 4 H11001 NH 4 H11001 H 2 C C COO H11002 CH 2 NAD(P)H H11001 H H11001 glutamate semialdehyde dehydrogenase NAD(P) H11001 COO H11002 Glutamate O H11001 N C HC N N C H C N H H FIGURE 18–26 Catabolic pathways for arginine, histidine, glutamate, glutamine, and proline. These amino acids are converted to H9251-keto- glutarate. The numbered steps in the histidine pathway are catalyzed by 1 histidine ammonia lyase, 2 urocanate hydratase, 3 imida- zolonepropionase, and 4 glutamate formimino transferase. 8885d_c18_656-689 2/3/04 11:39 AM Page 681 mac76 mac76:385_reb: of the carbon most distant from the carboxyl group to create a Schiff base, then hydrolysis of the Schiff base to a linear semialdehyde, glutamate H9253-semialdehyde. This intermediate is further oxidized at the same car- bon to produce glutamate. The action of glutaminase, or any of several enzyme reactions in which glutamine donates its amide nitrogen to an acceptor, converts glu- tamine to glutamate. Transamination or deamination of glutamate produces H9251-ketoglutarate. Arginine and histidine contain five adjacent car- bons and a sixth carbon attached through a nitrogen atom. The catabolic conversion of these amino acids to glutamate is therefore slightly more complex than the path from proline or glutamine (Fig. 18–26). Arginine is converted to the five-carbon skeleton of ornithine in the urea cycle (Fig. 18–10), and the ornithine is transami- nated to glutamate H9253-semialdehyde. Conversion of his- tidine to the five-carbon glutamate occurs in a multistep pathway; the extra carbon is removed in a step that uses tetrahydrofolate as cofactor. Four Amino Acids Are Converted to Succinyl-CoA The carbon skeletons of methionine, isoleucine, threo- nine, and valine are degraded by pathways that yield suc- cinyl-CoA (Fig. 18–27), an intermediate of the citric acid cycle. Methionine donates its methyl group to one of several possible acceptors through S-adenosylmethionine, Chapter 18 Amino Acid Oxidation and the Production of Urea682 CH HS H11002 OOC NH 3 H11001 CO 2 CO 2 CH 3 Cysteine Succinyl-CoA CHS NH 3 COO H11002 H11001 CH 3 CH 2 CH 2 3 steps Methionine Serine CH 2 CH 2 Homocysteine CH COO H11002 CH NH 3 H11001 Valine COO H11002 C O CH 2 CH 3 COO H11002 C O CH 2 NAD H11001 methylmalonyl- CoA mutase CoA-SH H9253-lyase coenzyme B 12 S-CoA Acetyl-CoA C O CH 2 CH 3 S-CoA H11002 6 steps HCO 3 Methylmalonyl-CoA CH 3 CH CH 2 CH H 2 O H11001 CH 3 CH OH Threonine COO H11002 2CO 2 CH 3 7 steps H11002 OOC C OCH 3 CH S-CoA COO H11002 CH 2 CH CH 3 NH 3 H11001 CH 3 C O S-CoA Propionyl-CoA Isoleucine NADH H11001 H H11001 H9251-keto acid dehydrogenase 2 steps threonine dehydratase PLP NH 3 H11001 NH 4 PLP PLP H9251-Ketobutyrate cystathionine cystathionine H9252-synthase FIGURE 18–27 Catabolic pathways for methionine, isoleucine, threonine, and valine. These amino acids are converted to succinyl- CoA; isoleucine also contributes two of its carbon atoms to acetyl-CoA (see Fig. 18–21). The pathway of threonine degradation shown here occurs in humans; a pathway found in other organisms is shown in Figure 18–19. The route from methionine to homocysteine is described in more detail in Figure 18–18; the conversion of homocysteine to H9251-ketobutyrate in Figure 22–14; the conversion of propionyl- CoA to succinyl-CoA in Figure 17–11. 8885d_c18_656-689 2/3/04 11:39 AM Page 682 mac76 mac76:385_reb: and three of its four remaining carbon atoms are con- verted to the propionate of propionyl-CoA, a precursor of succinyl-CoA. Isoleucine undergoes transamination, followed by oxidative decarboxylation of the resulting H9251- keto acid. The remaining five-carbon skeleton is further oxidized to acetyl-CoA and propionyl-CoA. Valine un- dergoes transamination and decarboxylation, then a se- ries of oxidation reactions that convert the remaining four carbons to propionyl-CoA. Some parts of the valine and isoleucine degradative pathways closely parallel steps in fatty acid degradation (see Fig. 17–8a). In human tissues, threonine is also converted in two steps to propionyl- CoA. This is the primary pathway for threonine degra- dation in humans (see Fig. 18–19 for the alternative pathway). The mechanism of the first step is analogous to that catalyzed by serine dehydratase, and the serine and threonine dehydratases may actually be the same enzyme. The propionyl-CoA derived from these three amino acids is converted to succinyl-CoA by a pathway de- scribed in Chapter 17: carboxylation to methylmalonyl- CoA, epimerization of the methylmalonyl-CoA, and conversion to succinyl-CoA by the coenzyme B 12 – dependent methylmalonyl-CoA mutase (see Fig. 17–11). In the rare genetic disease known as methylmalonic acidemia, methylmalonyl-CoA mutase is lacking—with serious metabolic consequences (Table 18–2; Box 18–2). Branched-Chain Amino Acids Are Not Degraded in the Liver Although much of the catabolism of amino acids takes place in the liver, the three amino acids with branched side chains (leucine, isoleucine, and valine) are oxidized as fuels primarily in muscle, adipose, kidney, and brain tissue. These extrahepatic tissues contain an amino- transferase, absent in liver, that acts on all three branched-chain amino acids to produce the correspon- ding H9251-keto acids (Fig. 18–28). The branched-chain H9251-keto acid dehydrogenase complex then catalyzes oxidative decarboxylation of all three H9251-keto acids, in each case releasing the carboxyl group as CO 2 and pro- ducing the acyl-CoA derivative. This reaction is formally analogous to two other oxidative decarboxylations encountered in Chapter 16: oxidation of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex (see Fig. 16–6) and oxidation of H9251-ketoglutarate to succinyl-CoA by the H9251-ketoglutarate dehydrogenase complex (p. 610). In fact, all three enzyme complexes are similar in structure and share essentially the same reaction mechanism. Five cofactors (thiamine pyro- phosphate, FAD, NAD, lipoate, and coenzyme A) par- ticipate, and the three proteins in each complex cat- alyze homologous reactions. This is clearly a case in which enzymatic machinery that evolved to catalyze 18.3 Pathways of Amino Acid Degradation 683 C CH CO 2 O CoA-SH S-CoA CH 3 CH 2 Valine Isoleucine Leucine H9251-Keto acids Acyl-CoA derivatives NAD branched-chain aminotransferase branched-chain H9251-keto acid dehydrogenase complex Maple syrup urine disease CH 3 C CHCH 3 CH 3 C CHCH 3 CH 2 CH 3 COO H11002 CH CH H11001 H 3 N CH 3 CH 2 CH 3 COO H11002 CH CH H11001 H 3 N CH 3 CH 3 COO H11002 CH CH H11001 H 3 N CH 3 CH 2 CH 3 COO H11002 C CHCH 3 CH 2 CH 3 COO H11002 C CHCH 3 CH 3 COO H11002 C CHCH 3 CH 2 CH 3 O O O S-CoAO S-CoAO FIGURE 18–28 Catabolic pathways for the three branched-chain amino acids: valine, isoleucine, and leucine. The three pathways, which occur in extrahepatic tissues, share the first two enzymes, as shown here. The branched-chain H9251-keto acid dehydrogenase complex is analogous to the pyruvate and H9251-ketoglutarate dehydrogenase com- plexes and requires the same five cofactors (some not shown here). This enzyme is defective in people with maple syrup urine disease. 8885d_c18_656-689 2/3/04 11:39 AM Page 683 mac76 mac76:385_reb: Chapter 18 Amino Acid Oxidation and the Production of Urea684 BOX 18–2 BIOCHEMISTRY IN MEDICINE Scientific Sleuths Solve a Murder Mystery Truth can sometimes be stranger than fiction—or at least as strange as a made-for-TV movie. Take, for ex- ample, the case of Patricia Stallings. Convicted of the murder of her infant son, she was sentenced to life in prison—but was later found innocent, thanks to the medical sleuthing of three persistent researchers. The story began in the summer of 1989 when Stallings brought her three-month-old son, Ryan, to the emergency room of Cardinal Glennon Children’s Hospital in St. Louis. The child had labored breathing, uncontrollable vomiting, and gastric distress. Accord- ing to the attending physician, a toxicologist, the child’s symptoms indicated that he had been poisoned with ethylene glycol, an ingredient of antifreeze, a conclusion apparently confirmed by analysis at a commercial lab. After he recovered, the child was placed in a fos- ter home, and Stallings and her husband, David, were allowed to see him in supervised visits. But when the infant became ill, and subsequently died, after a visit in which Stallings had been briefly left alone with him, she was charged with first-degree murder and held without bail. At the time, the evidence seemed com- pelling as both the commercial lab and the hospital lab found large amounts of ethylene glycol in the boy’s blood and traces of it in a bottle of milk Stallings had fed her son during the visit. But without knowing it, Stallings had performed a brilliant experiment. While in custody, she learned she was pregnant; she subsequently gave birth to an- other son, David Stallings Jr., in February 1990. He was placed immediately in a foster home, but within two weeks he started having symptoms similar to Ryan’s. David was eventually diagnosed with a rare metabolic disorder called methylmalonic acidemia (MMA). A recessive genetic disorder of amino acid metabolism, MMA affects about 1 in 48,000 newborns and presents symptoms almost identical with those caused by ethylene glycol poisoning. Stallings couldn’t possibly have poisoned her sec- ond son, but the Missouri state prosecutor’s office was not impressed by the new developments and pressed forward with her trial anyway. The court wouldn’t al- low the MMA diagnosis of the second child to be in- troduced as evidence, and in January 1991 Patricia Stallings was convicted of assault with a deadly weapon and sentenced to life in prison. Fortunately for Stallings, however, William Sly, chairman of the Department of Biochemistry and Mol- ecular Biology at St. Louis University, and James Shoemaker, head of a metabolic screening lab at the university, got interested in her case when they heard about it from a television broadcast. Shoemaker per- formed his own analysis of Ryan’s blood and didn’t de- tect ethylene glycol. He and Sly then contacted Piero Rinaldo, a metabolic disease expert at Yale University School of Medicine whose lab is equipped to diagnose MMA from blood samples. When Rinaldo analyzed Ryan’s blood serum, he found high concentrations of methylmalonic acid, a breakdown product of the branched-chain amino acids isoleucine and valine, which accumulates in MMA pa- tients because the enzyme that should convert it to the next product in the metabolic pathway is defec- tive. And particularly telling, he says, the child’s blood and urine contained massive amounts of ketones, an- other metabolic consequence of the disease. Like Shoemaker, he did not find any ethylene glycol in a sample of the baby’s bodily fluids. The bottle couldn’t be tested, since it had mysteriously disappeared. Ri- naldo’s analyses convinced him that Ryan had died from MMA, but how to account for the results from two labs, indicating that the boy had ethylene glycol in his blood? Could they both be wrong? When Rinaldo obtained the lab reports, what he saw was, he says, “scary.” One lab said that Ryan Stallings’ blood contained ethylene glycol, even though the blood sample analysis did not match the lab’s own profile for a known sample containing eth- ylene glycol. “This was not just a matter of question- able interpretation. The quality of their analysis was unacceptable,” Rinaldo says. And the second labora- tory? According to Rinaldo, that lab detected an ab- normal component in Ryan’s blood and just “assumed it was ethylene glycol.” Samples from the bottle had produced nothing unusual, says Rinaldo, yet the lab claimed evidence of ethylene glycol in that, too. Rinaldo presented his findings to the case’s pros- ecutor, George McElroy, who called a press confer- ence the very next day. “I no longer believe the labo- ratory data,” he told reporters. Having concluded that Ryan Stallings had died of MMA after all, McElroy dismissed all charges against Patricia Stallings on September 20, 1991. By Michelle Hoffman (1991). Science 253, 931. Copyright 1991 by the American Association for the Advancement of Science. 8885d_c18_656-689 2/3/04 11:39 AM Page 684 mac76 mac76:385_reb: 18.3 Pathways of Amino Acid Degradation 685 one reaction was “borrowed” by gene duplication and further evolved to catalyze similar reactions in other pathways. Experiments with rats have shown that the branched-chain H9251-keto acid dehydrogenase complex is regulated by covalent modification in response to the content of branched-chain amino acids in the diet. With little or no excess dietary intake of branched-chain amino acids, the enzyme complex is phosphorylated and thereby inactivated by a protein kinase. Addition of ex- cess branched-chain amino acids to the diet results in dephosphorylation and consequent activation of the en- zyme. Recall that the pyruvate dehydrogenase complex is subject to similar regulation by phosphorylation and dephosphorylation (p. 621). There is a relatively rare genetic disease in which the three branched-chain H9251-keto acids (as well as their precursor amino acids, especially leucine) ac- cumulate in the blood and “spill over” into the urine. This condition, called maple syrup urine disease be- cause of the characteristic odor imparted to the urine by the H9251-keto acids, results from a defective branched- chain H9251-keto acid dehydrogenase complex. Untreated, the disease results in abnormal development of the brain, mental retardation, and death in early infancy. Treatment entails rigid control of the diet, limiting the intake of valine, isoleucine, and leucine to the minimum required to permit normal growth. ■ Asparagine and Aspartate Are Degraded to Oxaloacetate The carbon skeletons of asparagine and aspartate ultimately enter the citric acid cycle as oxaloacetate. The enzyme asparaginase catalyzes the hydrolysis of asparagine to aspartate, which undergoes transamina- tion with H9251-ketoglutarate to yield glutamate and oxalo- acetate (Fig. 18–29). We have now seen how the 20 common amino acids, after losing their nitrogen atoms, are degraded by dehydrogenation, decarboxylation, and other reactions to yield portions of their carbon backbones in the form of six central metabolites that can enter the citric acid cycle. Those portions degraded to acetyl-CoA are completely oxidized to carbon dioxide and water, with generation of ATP by oxidative phosphorylation. As was the case for carbohydrates and lipids, the degradation of amino acids results ultimately in the gen- eration of reducing equivalents (NADH and FADH 2 ) through the action of the citric acid cycle. Our survey of catabolic processes concludes in the next chapter with a discussion of respiration, in which these reduc- ing equivalents fuel the ultimate oxidative and energy- generating process in aerobic organisms. FIGURE 18–29 Catabolic pathway for asparagine and aspartate. Both amino acids are converted to oxaloacetate. CCH 2 COO H11002 H11001 H 2 O C O O asparaginase aspartate aminotransferase H9251-Ketoglutarate PLP H11002 O Asparagine Aspartate Oxaloacetate CCHCH 2 NH 3 COO H11002 H11001 O H 2 N CCHCH 2 NH 4 COO H11002 O H11002 O Glutamate H11001 NH 3 SUMMARY 18.3 Pathways of Amino Acid Degradation ■ After removal of their amino groups, the carbon skeletons of amino acids undergo oxidation to compounds that can enter the citric acid cycle for oxidation to CO 2 and H 2 O. The reactions of these pathways require a number of cofactors, including tetrahydrofolate and S-adenosylmethionine in one-carbon transfer reactions and tetrahydrobiopterin in the oxidation of phenylalanine by phenylalanine hydroxylase. ■ Depending on their degradative end product, some amino acids can be converted to ketone bodies, some to glucose, and some to both. Thus amino acid degradation is integrated into intermediary metabolism and can be critical to survival under conditions in which amino acids are a significant source of metabolic energy. ■ The carbon skeletons of amino acids enter the citric acid cycle through five intermediates: acetyl-CoA, H9251-ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate. Some are also degraded to pyruvate, which can be converted to either acetyl-CoA or oxaloacetate. 8885d_c18_656-689 2/3/04 11:39 AM Page 685 mac76 mac76:385_reb: ■ The amino acids producing pyruvate are alanine, cysteine, glycine, serine, threonine, and tryptophan. Leucine, lysine, phenylalanine, and tryptophan yield acetyl-CoA via acetoacetyl-CoA. Isoleucine, leucine, threonine, and tryptophan also form acetyl-CoA directly. ■ Arginine, glutamate, glutamine, histidine, and proline produce H9251-ketoglutarate; isoleucine, methionine, threonine, and valine produce succinyl-CoA; four carbon atoms of phenylalanine and tyrosine give rise to fumarate; and asparagine and aspartate produce oxaloacetate. ■ The branched-chain amino acids (isoleucine, leucine, and valine), unlike the other amino acids, are degraded only in extrahepatic tissues. ■ A number of serious human diseases can be traced to genetic defects in the enzymes of amino acid catabolism. Chapter 18 Amino Acid Oxidation and the Production of Urea686 Key Terms aminotransferases 660 transaminases 660 transamination 660 pyridoxal phosphate (PLP) 660 oxidative deamination 661 L-glutamate dehydrogenase 661 glutamine synthetase 662 glutaminase 663 creatine kinase 664 glucose-alanine cycle 664 ammonotelic 665 ureotelic 665 uricotelic 665 urea cycle 665 urea 668 essential amino acids 669 ketogenic 672 glucogenic 672 tetrahydrofolate 672 S-adenosylmethionine (adoMet) 672 tetrahydrobiopterin 674 phenylketonuria (PKU) 679 mixed-function oxidases 679 alkaptonuria 681 maple syrup urine disease 685 Terms in bold are defined in the glossary. Further Reading General Arias, I.M., Boyer, J.L., Chisari, F.V., Fausto, N., Schachter, D., & Shafritz, D.A. (2001) The Liver: Biology and Pathobiology, 4th edn, Lippincott Williams & Wilkins, Philadelphia. Bender, D.A. (1985) Amino Acid Metabolism, 2nd edn, Wiley- Interscience, Inc., New York. Brosnan, J.T. (2001) Amino acids, then and now—a reflection on Sir Hans Krebs’ contribution to nitrogen metabolism. IUBMB Life 52, 265–270. An interesting tour through the life of this important biochemist. Campbell, J.W. (1991) Excretory nitrogen metabolism. In Environmental and Metabolic Animal Physiology, 4th edn (Prosser, C.L., ed.), pp. 277–324, John Wiley & Sons, Inc., New York. Coomes, M.W. (1997) Amino acid metabolism. In Textbook of Biochemistry with Clinical Correlations, 5th edn (Devlin, T.M., ed.), pp. 779–823, Wiley-Liss, New York. Hayashi, H. (1995) Pyridoxal enzymes: mechanistic diversity and uniformity. J. Biochem. 118, 463–473. Mazelis, M. (1980) Amino acid catabolism. In The Biochemistry of Plants: A Comprehensive Treatise (Stumpf, P.K. & Conn, E.E., eds), Vol. 5: Amino Acids and Derivatives (Miflin, B.J., ed.), pp. 541–567, Academic Press, Inc., New York. A discussion of the various fates of amino acids in plants. Walsh, C. (1979) Enzymatic Reaction Mechanisms, W. H. Freeman and Company, San Francisco. A good source for in-depth discussion of the classes of enzymatic reaction mechanisms described in the chapter. Amino Acid Metabolism Christen, P. & Metzler, D.E. (1985) Transaminases, Wiley- Interscience, Inc., New York. Curthoys, N.P. & Watford, M. (1995) Regulation of glutaminase activity and glutamine metabolism. Annu. Rev. Nutr. 15, 133–159. Fitzpatrick, P.F. (1999) Tetrahydropterin-dependent amino acid hydroxylases. Annu. Rev. Biochem. 68, 355–382. Kirsch, J.F. & Eliot, A.C. (2004) Pyridoxal phosphate enzymes: mechanistic, structural and evolutionary considerations. Annu. Rev. Biochem. 73 [in press]. Pencharz, P.B. & Ball, R.O. (2003) Different approaches to define individual amino acid requirements. Annu. Rev. Nutr. 23, 101–116. Determination of which amino acids are essential in the human diet is not a trivial problem, as this review relates. The Urea Cycle Brusilow, S.W. & Horwich, A.L. (2001) Urea cycle enzymes. In The Metabolic Bases of Inherited Disease, 8th edn (Scriver, C.R., Beaudet, A.C., Sly, W.S., Valle, D., Childs, B., Kinzler, K., & Vogelstein, B., eds), pp. 1909–1963, McGraw-Hill Companies Inc., New York. An authoritative source on this pathway. 8885d_c18_686 2/3/04 4:14 PM Page 686 mac76 mac76:385_reb: Chapter 18 Problems 687 Holmes, F.L. (1980) Hans Krebs and the discovery of the ornithine cycle. Fed. Proc. 39, 216–225. A medical historian reconstructs the events leading to the discovery of the urea cycle. Kirsch, J.F., Eichele, G., Ford, G.C., Vincent, M.G., Jansonius, J.N., Gehring, H., & Christen, P. (1984) Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J. Mol. Biol. 174, 497–525. Morris, S.M. (2002) Regulation of enzymes of the urea cycle and arginine metabolism. Annu. Rev. Nutr. 22, 87–105. This review details what is known about some levels of regulation not covered in the chapter, such as hormonal and nutritional regulation. Disorders of Amino Acid Degradation Ledley, F.D., Levy, H.L., & Woo, S.L.C. (1986) Molecular analysis of the inheritance of phenylketonuria and mild hyperphenylalaninemia in families with both disorders. N. Engl. J. Med. 314, 1276–1280. Nyhan, W.L. (1984) Abnormalities in Amino Acid Metabolism in Clinical Medicine, Appleton-Century-Crofts, Norwalk, CT. Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D., Childs, B., Kinzler, A.W., & Vogelstein, B. (eds) (2001) The Metabolic and Molecular Bases of Inherited Disease, 8th edn, Part 5: Amino Acids, McGraw-Hill, Inc., New York. Scriver, C.R., Kaufman, S., & Woo, S.L.C. (1988) Mendelian hyperphenylalaninemia. Annu. Rev. Genet. 22, 301–321. 1. Products of Amino Acid Transamination Name and draw the structure of the H9251-keto acid resulting when each of the following amino acids undergoes transamination with H9251-ketoglutarate: (a) aspartate, (b) glutamate, (c) alanine, (d) phenylalanine. 2. Measurement of Alanine Aminotransferase Activ- ity The activity (reaction rate) of alanine aminotransferase is usually measured by including an excess of pure lactate de- hydrogenase and NADH in the reaction system. The rate of alanine disappearance is equal to the rate of NADH disap- pearance measured spectrophotometrically. Explain how this assay works. 3. Distribution of Amino Nitrogen If your diet is rich in alanine but deficient in aspartate, will you show signs of aspartate deficiency? Explain. 4. A Genetic Defect in Amino Acid Metabolism: A Case History A two-year-old child was taken to the hospital. His mother said that he vomited frequently, especially after feedings. The child’s weight and physical development were below normal. His hair, although dark, con- tained patches of white. A urine sample treated with ferric chloride (FeCl 3 ) gave a green color characteristic of the pres- ence of phenylpyruvate. Quantitative analysis of urine sam- ples gave the results shown in the table. (a) Suggest which enzyme might be deficient in this child. Propose a treatment. (b) Why does phenylalanine appear in the urine in large amounts? (c) What is the source of phenylpyruvate and phenyl- lactate? Why does this pathway (normally not functional) come into play when the concentration of phenylalanine rises? (d) Why does the boy’s hair contain patches of white? 5. Role of Cobalamin in Amino Acid Catabolism Pernicious anemia is caused by impaired absorption of vitamin B 12 . What is the effect of this impairment on the catabolism of amino acids? Are all amino acids equally af- fected? (Hint: See Box 17–2.) 6. Lactate versus Alanine as Metabolic Fuel: The Cost of Nitrogen Removal The three carbons in lactate and ala- nine have identical oxidation states, and animals can use ei- ther carbon source as a metabolic fuel. Compare the net ATP yield (moles of ATP per mole of substrate) for the complete oxidation (to CO 2 and H 2 O) of lactate versus alanine when the cost of nitrogen excretion as urea is included. 7. Pathway of Carbon and Nitrogen in Glutamate Metabolism When [2- 14 C, 15 N] glutamate undergoes oxi- dative degradation in the liver of a rat, in which atoms of the following metabolites will each isotope be found: (a) urea, (b) succinate, (c) arginine, (d) citrulline, (e) ornithine, (f) aspartate? CH 2 H COO H11002 H H C Labeled glutamate COO H11002 15 14 CH 2 H N H11001 A O COO H11002 O O C Lactate A A H HO OH HH C A H11001 O COO H11002 O O C A A H OH HH C Alanine H 3 N Concentration (mM) Substance Patient’s urine Normal urine Phenylalanine 7.0 0.01 Phenylpyruvate 4.8 0 Phenyllactate 10.3 0 Problems 8885d_c18_656-689 2/3/04 11:39 AM Page 687 mac76 mac76:385_reb: Chapter 18 Amino Acid Oxidation and the Production of Urea688 8. Chemical Strategy of Isoleucine Catabolism Isoleucine is degraded in six steps to propionyl-CoA and acetyl-CoA: (a) The chemical process of isoleucine degradation in- cludes strategies analogous to those used in the citric acid cycle and the H9252 oxidation of fatty acids. The intermediates of isoleucine degradation (I to V) shown below are not in the proper order. Use your knowledge and understanding of the citric acid cycle and H9252-oxidation pathway to arrange the in- termediates in the proper metabolic sequence for isoleucine degradation. (b) For each step you propose, describe the chemical process, provide an analogous example from the citric acid cycle or H9252-oxidation pathway (where possible), and indicate any necessary cofactors. 9. Role of Pyridoxal Phosphate in Glycine Metabolism The enzyme serine hydroxymethyltransferase requires pyri- doxal phosphate as cofactor. Propose a mechanism for the re- action catalyzed by this enzyme, in the direction of serine degradation (glycine production). (Hint: See Figs 18–19 and 18–20b.) 10. Parallel Pathways for Amino Acid and Fatty Acid Degradation The carbon skeleton of leucine is degraded by a series of reactions closely analogous to those of the cit- ric acid cycle and H9252 oxidation. For each reaction, (a) through (f), indicate its type, provide an analogous example from the citric acid cycle or H9252-oxidation pathway (where possible), and note any necessary cofactors. CH 2 H S-CoAO CH 3 C CCH 3 III IV V C C OO H11002 OC CH 3 HCH 3 CH 2 H CH 3 C S-CoA CH 3 C C O III C S-CoAO CH 3 C CCH 3 O H C S-CoA CH 3 C C O H H CH 3 HO C C H CH 2 Isoleucine Propionyl-CoACH 3 H11001 H 3 N OO H11002 C S-CoA CH 3 CH 3 O C H CH 3 6 steps C S-CoA CH 2 O H11001 Acetyl-CoA H11002 OOC CH 3 NH 3 CH 2 O CO 2 H11001 CCH 3 Acetyl-CoA (b) CH 3 COO H11002 CH 2 C C H H 2 O Leucine (c) CoA-SH CH 3 COO H11002 CH 2 C O C H CH 3 H9251-Ketoisocaproate (e) S-CoA CH 3 CH 2 C O C CH 3 Isovaleryl-CoA S-CoA C H (f ) H C C C O S-CoA H H9252-Methylcrotonyl-CoA (d) H11002 OOC CCH 2 O C H 3 C H9252-Methylglutaconyl-CoA C C S-CoA H H11002 OOC CH 2 O C CH 3 H9252-Hydroxy-H9252-methylglutaryl-CoA C S-CoA (a) CH 2 OH CH 3 O Acetoacetate CH 3 H11002 HCO 3 H 3 H11001 8885d_c18_656-689 2/3/04 11:39 AM Page 688 mac76 mac76:385_reb: Chapter 18 Problems 689 11. Ammonia Toxicity Resulting from an Arginine- Deficient Diet In a study conducted some years ago, cats were fasted overnight then given a single meal complete in all amino acids except arginine. Within 2 hours, blood am- monia levels increased from a normal level of 18 H9262g/L to 140 H9262g/L, and the cats showed the clinical symptoms of ammo- nia toxicity. A control group fed a complete amino acid diet or an amino acid diet in which arginine was replaced by or- nithine showed no unusual clinical symptoms. (a) What was the role of fasting in the experiment? (b) What caused the ammonia levels to rise in the ex- perimental group? Why did the absence of arginine lead to ammonia toxicity? Is arginine an essential amino acid in cats? Why or why not? (c) Why can ornithine be substituted for arginine? 12. Oxidation of Glutamate Write a series of balanced equations, and an overall equation for the net reaction, de- scribing the oxidation of 2 mol of glutamate to 2 mol of H9251- ketoglutarate and 1 mol of urea. 13. Transamination and the Urea Cycle Aspartate aminotransferase has the highest activity of all the mam- malian liver aminotransferases. Why? 14. The Case against the Liquid Protein Diet A weight-reducing diet heavily promoted some years ago required the daily intake of “liquid protein” (soup of hy- drolyzed gelatin), water, and an assortment of vitamins. All other food and drink were to be avoided. People on this diet typically lost 10 to 14 lb in the first week. (a) Opponents argued that the weight loss was almost entirely due to water loss and would be regained very soon after a normal diet was resumed. What is the biochemical ba- sis for this argument? (b) A number of people on this diet died. What are some of the dangers inherent in the diet, and how can they lead to death? 15. Alanine and Glutamine in the Blood Normal human blood plasma contains all the amino acids required for the synthesis of body proteins, but not in equal concentrations. Alanine and glutamine are present in much higher concen- trations than any other amino acids. Suggest why. 8885d_c18_656-689 2/3/04 11:39 AM Page 689 mac76 mac76:385_reb: