? C1 Introduction to enzymes
? C2 Thermodynamics热力学
? C3 Enzyme kinetics动力学
? C4 Enzyme inhibition
? C5 Regulation of enzyme
activity
C Enzymes
? Enzyme as catalysts催化剂
? Active site 活性部位
? Substrate specificity底物专一性
? Enzyme classification分类
? Enzyme assays酶的测定
? Linked enzyme assays
? Coenzymes辅酶 and prosthetic groups辅基
? Isoenzymes同工酶
C1 Introduction to enzymes
Enzymes as catalysts
? Enzymes are catalysts that change the rate of a
reaction without being changed themselves
? Enzyme catalyzed reactions usually take place under
relatively mild conditions(T,P,pH),
? Enzymes are highly specific and their activity can be
regulated,
? Virtually all enzymes are proteins,although some
catalytically active RNAs have been identified,
Active site活性部位
? Active site is the region that binds the substrate 底物
and converts it into product。
? 结合部位：负责与底物的结合，决定酶的专一性。
? 催化部位：负责催化底物键的断裂形成新键，决定酶的催
化能力。
? Small（ 1-2%）,a three-dimensional entity实体
? Cleft or crevice裂缝 on the surface of enzyme
? Bind by multiple weak forces
? Enzyme-substrate complex
? transition state complex过渡态复合物 --?product
Active site
Active site
Enzyme-substrate complex
Two models---enzyme bind its
substrate
? Lock and key model锁 -钥,Emil Fischer,1894
? Induced fit model诱导契合,Daniel E,
Koshland,Jr 1958
Lock and key model
Induced fit model
Induced fit model
? Active sites in the uninduced enzyme are shown schematically with
round contours,Binding of the first substrate (gold) induces a
conformational shift (angular contours) that facilitates binding of the
second substrate (blue),with far lower energy than otherwise
required,When catalysis is complete,the product is released,and
the enzyme returns to its uninduced state,
Substrate specificity
? Substrate specificity is often determined by
changes in relatively few amino acids in the
active site,
? 结构专一性,
1) 绝对专一性（脲酶）；
2) 相对专一性（酶作用于一类结构相近的底物）
? 立体异构专一性
1) 旋光异构专一性（ L-氨基酸氧化酶）
2) 几何异构专一性
Serine proteases丝氨酸蛋白酶家族
? Trypsin胰蛋白酶 cleaves on the carboxyl side
of positively charged Lys or Arg residues,
? Chymotrypsin 胰凝乳蛋白酶, cleaves on the
carboxyl side of bulky aromatic and
hydrophobic amino acid residues
? Elastase 弹性蛋白酶, cleaves on the carboxyl
side of small uncharged side chains
COO
-
C
A s p
COO
-
C
A s p
T r y p sin Ch y m o tr y p sin E last a se
切 L y s,Ar g 切 T rp,Phe,T y r 切 Ala,Gl y
Non - p o l a r
p o ck e t
Dee
p
a
n
d
n
e
g
a
tiv
e
l
y
ch
a
r
g
e
d
p
o
ck
e
t
S h a l l o w a n d
n o n - p o l a r
p o ck e t
O O
– C – N – C – C – N –
C
C
C
C
NH
3
+
O O
– C – N – C – C – N –
C
O O
– C – N – C – C – N –
CH
3
Ju
a
n
g
R
H
(2
0
0
4
)
BC
b
a
si
cs
Enzyme classification
2,Enzyme classification
? 氧化 -还原酶催化氧化 -还原反应。
? 主要包括脱氢酶 (dehydrogenase)和氧化酶 (Oxidase)。
? 如，乳酸 (Lactate)脱氢酶催化乳酸的脱氢反应。
(1) 氧化 -还原酶 Oxidoreductase
C H 3 C H C O O H
O H
N A D + H +C H 3 C C O O H
O
N A D H
2,Enzyme classification
? 转移酶催化基团转移反应，即将一个底物分子的基团或原子转移到
另一个底物的分子上。
例如,谷丙转氨酶催化的氨基转移反应。
(2) 转移酶 Transferase
C H 3 C H C O O H
N H 2
H O O C C H 2 C H 2 C C O O H
O
H O O C C H 2 C H 2 C H C O O H
N H 2
C H 3 C C O O H
O
2,Enzyme classification
? 水解酶催化底物的加水分解反应。
? 主要包括淀粉酶、蛋白酶、核酸酶及脂酶等。
? 例如，脂肪酶 (Lipase)催化的脂的水解反应,
(3) 水解酶 hydrolase
H 2 OC O O C H 2 C H 3R R C O O H C H 3 C H 2 O H
Enzyme classification
? 裂合酶催化从底物分子中移去一个基团或原子形成双键
的反应及其逆反应。
? 主要包括醛缩酶、水化酶及脱氨酶等。
? 例如,延胡索酸水合酶催化的反应。
(4) 裂合酶 Lyase
H O O C C H = C H C O O H H 2 O H O O C C H 2 C H C O O H
O H
Enzyme classification
? 异构酶催化各种同分异构体的相互转化，即底物分子内
基团或原子的重排过程。
例如,6-磷酸葡萄糖异构酶催化的反应。
(5) 异构酶 Isomerase
O
C H 2 O H
O H
O H
O H
O H O
C H 2 O H C H 2 O H
O H
O H
O H
Enzyme classification
? 合成酶，又称为连接酶，能够催化 C-C,C-O,C-N 以及
C-S 键的形成反应。这类反应必须与 ATP分解反应相互
偶联。
? A + B + ATP + H-O-H ===A ? B + ADP +Pi
? 例如，丙酮酸羧化酶催化的反应。
丙酮酸 + CO2 ? 草酰乙酸
(6) 合成酶 Ligase or Synthetase
Enzyme classification
? 核酸酶是唯一的非蛋白酶。它是一类特殊的 RNA，能够催化 RNA分子
中的磷酸酯键的水解及其逆反应。
(7) 核酸酶（催化核酸） ribozyme
3'3'
3'3'3'
3'3'3'3'
3'
5'5'5'5'
5'
5'5'5'5'5'
BBBBB
PP P
+
P O HPP
BBBB
PPPP
P
B
P
Enzyme classification
? Trypsin’s Enzyme Commission number is 3.4.21.4
? 3 denote it is a hydrolase
? 4 denote it is a protease that hydrolyzes peptide
bonds
? 21 denote it is a serine protease with a serine
residue at the active site
? 4 indicates that it was the fourth enzyme to be
assigned to this class,
Enzyme assays
? The amount of enzyme protein present can be
determined in terms of the catalytic effect it
produces,that is the conversion of substrate
to product,
1,The overall equation of the reaction
2,The disappearance of substrate or the
appearance of product
3,Cofactors,pH,temperature
4,Sufficient supply of substrate
Enzyme assay
? Enzyme activity（酶催化某一化学反应的能
力）,the rate of appearance of product or the
rate of disappearance of substrate
? Absorbance:spectrophotometer分光光度计
? Two most common molecules,
? NADH(reduced nicotinamide ademine
dinucleotied还原型烟酰胺腺嘌呤二核苷酸 )
? NADPH:340nm
NADH (NADPH)
LDH:Lactate dehydrogenase
Linked enzyme assays
? If neither the substrates nor products of
an enzyme-catalyzed reaction absorb light
at an appropriate wavelength,the enzyme
can be assayed by linking to another
enzyme-catalyzed reaction that does
involve a change in absorbance,
? The second enzyme must be in excess,so
that the rate-limit step in the linked assay
is the action of the first enzyme,
Coenzymes and prosthetic groups
? Cofactors:small,nonprotein units(iorganic ions or a
complex organic molecule called coenzyme辅酶 )
? Prosthetic group 辅基,A metal or coenzyme that is
covalently attached to the enzyme is called a
prosthetic group,
? Holoenzyme全酶 =cofactor辅因子 +apoenzyme脱辅酶
? Many coenzymes are derived from vitamin
precursors,giving rise to deficiency diseases when
in inadequate supply,
? P73 table 2
Cofactors
NAD+（辅酶 I） and NADP+(辅酶 II)
? NAD+ and NADP+ have a common function as they
both act as carriers of electrons and are involved in
oxydation-reduction reactions,
? NAD+ is more commonly used in catabolic(break
down)分解代谢 reactions
? NADP+ is used in anabolic(biosynthetic)合成代谢
reactions,
? The reactive part of both molecules is the
nicotinamide ring which exists in reduced or an
oxidized form,and so acts to accept or donate
electrons in an enzyme reaction,
? NAD+ +H++2e- < ----- > NADH
NAD+
NADP
FAD and FMN
? Flavin adenine dinucleotide(FAD) 黄素腺嘌呤二核 苷
酸 and flavin mononucleotide(FMN) 黄素单核苷酸 are
also carriers of electrons,
? Reactive site is flavine mononucleotide unit,
? FAD and FMN react with two protons as well as two
electrons,in alternating between the reduced and
oxidized state,
? FAD+2H++2e-< ----- >FADH2
FMN
FAD
Isoenzymes:同工酶
? Isoenzymes are different forms of an
enzyme which catalyze the same
reaction,but which exhibit different physical
or kinetic properties(pI,pH,substrate
affinity or inhibitors)
? LDH:乳酸脱氢酶
? CH3CH(OH)COO-+NAD+< ---->CH3COCOO-
+NADH+H+
LDH
? LDH is a tetramer of two different types of
subunits,called H and M,which have small
differences in amino acid sequence,
? The two subunits can combine randomly with each
other,forming 5 isoenzymes that have the
compositions H4,H3M,H2M2,HM3,M4,
? They can be resolved electrophoretically,
? M subnunits predominate in skeletal muscle and
liver,whereas H subunits predominate in the heart,
? H4,H3M:heart and red blood cells
? H2M2:brain and kidney
? HM3,M4,liver and skeletal muscle
C2 Thermodynamics
1,Thermodynamics热力学
2,Activation energy 活化能 and transition
state
3,Free energy change
4,Chemical equilibria化学平衡
Thermodynamics
? A knowledge of thermodynamics,(which is the
description among the various forms of energy and
how energy affects matter),enables one to determine
whether a physical process is possible,
? In Thermodynamics,a system is the matter within a
defined region,
? The matter in the rest of the universe is called the
surroundings,
The first laws of thermodynamics
? ΔE=EA-EB=Q-W
? EA is the energy of the system at the start of a
process
? EB is the energy of the system at the end of the
process
? Q is the heat absorbed by the system
? W is the work done by the system,
? Q = ΔE+ W 系统从外界吸收的热量等于系统内能
的增量和系统对外界做功之和 。这就是热力学第
一定律。
The second laws of thermodynamics
? The second laws of thermodynamics states that a
process can occur spontaneously only if the sum of
the entropies 熵 (Entropy is a measure of the degree
of randomness or disorder of a system)of the
system and its surroundings increases,
? ΔS system+ Δ S surroundings >0 for a spontaneous
process
? Entropy changes of chemical reactions are not
readily measured,
Free energy自由能 (G)
? Josiah Willard Gibbs
? The first and second laws of thermodynamics are
combined in the thermodynamic function,free
energy,
? ΔG= ΔH-T ΔS(ΔH:热量的变化； ΔS：熵变 )
? ΔH= ΔE+P ΔV
? ΔG= ΔE-T ΔS
? If △ G is negative,that reaction can happen
spontaneously
? If △ G is positive,an input of energy is required to
drive the reaction,
Activation energy and transition state
For a biochemical reaction to proceed,the
energy barrier needed to transform the
substrate molecules into the transition state
has to overcome,
The transition state has the highest free energy in
the energy pathway,
Gibbs 活化自由能
ΔG++（ Gibbs 活化自由能 ）,
The difference in free
energy between the
substrate and
transition state is
termed the Gibbs free
energy of activation,
△ G ++
An enzyme stabilizes the
transition state and
lowers △ G ++,thus
increasing the rate at
which the reaction
occurs,
△ G ++ and △ G
△ Go’
? ATP+H2O------->ADP+Pi
? △ Go’=-30.5kJ mol-1=-7.3kcalmol-1
? 1kcal卡 =4.184kJ
Chemical equilibria
? A chemical reaction often exists in a state of dynamic
equilibrium,
? The equilibrium constant ( K) defines the ratio of the substrates
and product at equilibrium,
? Enzymes do not alter the equilibrium position,but do accelerate
the attainment of the equilibrium position by speeding up the
forward and reverse reactions,
C3 Enzyme kinetics
? Enzyme velocity
? Substrate Concentration
? Enzyme concentration
? Temperature
? pH
? Michaelis-menten Model
? Lineweaver-Burk Plot
Enzyme velocity
? Enzyme activity is commonly expressed by the
intial rate ( V0) of the reaction being catalyzed,
The units of V 0are u mol/ min。 (why?)
Enzyme unit
? Enzyme activity may be expressed in a number of
ways,the commonest is by the V0
? There are also 2 standard units of enzyme activity,
? the enzyme unit (U) and the katal(kat),
? 1U,the amount of enzyme which will catalyze the
transformation of 1 u mol of substrate per min at
25oC under optimal conditions,
? 1kat:在最适条件下，每秒钟能催化 1mol底物转化为产物
所需的酶量。
? 1U=16.67 nkat,
Specific activity比活力
The specific activity is the number of units per
milligram of protein(units /mg)
Or 每 g酶制剂或每 ml酶制剂含有多少个活力单位表
示（ U/g,U/ml）。用来比较每单位质量蛋白质的
催化能力。
? The specific activity is a measure of the
purity of an enzyme,
Substrate Concentration
? At low substrate concentrations,a doubling of [S]
leads to a doubling of V 。,whereas at higher [S] the
enzyme becomes saturated and there is no further
increase in V 。
Substrate Concentration
1,中间复合物学说
2,饱和现象
3,双曲线
Enzyme concentration
? When [S] is saturating,a doubling of the
enzyme concentration leads to a
doubling of V0,
Temperature
? Temperature affects the rate of an enzyme-catalyzed
reaction by increasing the thermal energy of the
substrate molecules,
? This increases the proportion of molecules with
sufficient energy to overcome the activation barrier
and hence increases the rate of the reaction,
? in addition,the thermal energy of the component
molecules of the enzyme is increased,which leads to
an increased rate of denaturation of the enzyme
protein due to the disruption of the noncovalent
interactions holding the structure together,
T
pH
? Each enzyme has an optimum pH at which the rate of
the reaction that it catalyzes is at its maximum,
? Slight deviations in the pH from the optimum lead to
a decrease in the reaction rate,
? Larger deviations in pH lead to denaturation of the
enzyme due to charges in the ionization of amino
acid residues and the disruption of noncovalent
interactions
pH
Michaelis-menten Model
Vmax指该酶促反应的最大速度,[S]为底物浓度,Km是米氏常数,VO是在
某一底物浓度时相应的反应速度。
Michaelis-Menten model
Michaelis-Menten model
Michaelis-Menten model
[E]+[S]------->[ES] k1
[ES]-----> [E]+[S] K-1
[ES]------- >[E]+[P] k2
Michaelis-Menten model
Michaelis-Menten model
? The concentration of uncombined enzyme [E] is equal
? to the total enzyme concentration [E]T minus the
concentration of the [ES]complex,
Michaelis-Menten model
Michaelis-Menten model
? The maximal rate,Vmax,is attained when the catalytic
sites on the enzyme are saturated with substrate— that is,
when [ES] = [E]T,Thus,
A direct plot
当反应速度等于
最大速度一半时,
即 V = 1/2 Vmax,
Km = [S]
Km
Kcat:催化常数，转换数,每秒钟每个酶
分子转换底物的分子数。
A lineweaver-burk double-
reciprocal plot
C4 Enzyme inhibition
1,Enzyme inhibition
2,Irreversible inhibition
3,Reversible competitive inhibition
4,Reversible noncompetitive inhibition
Enzyme inhibition
? Inhibitor:any molecule which acts directly
on an enzyme to lower its catalytic rate is
called an inhibitor.(not denaturation)
? Some enzyme inhibitors are normal body
metabolites,
? other may be foreign substances,such as
drugs or toxins
Irreversible
? Irreversible:An irreversible inhibitor binds
tightly,often covalently,to amino acid
residues at the active site of the enzyme,
permanently inactivating the enzyme,
1.非专一性不可逆抑制
2.专一性不可逆抑制
diisopropylfluorophosphate( DIPF)
Iodoacetamide 碘乙酰胺
Antibiotic penicillin
Penicillin
Irreversible
e.g
Reversible Competitive inhibition
A competitive inhibitor
typically has colse
structural similarities to
the normal substrate for
the enzyme,
Thus it competes with the
substrate molecules for
binding to the active
site of the enzyme,
Reversible Competitive inhibition
? At high substrate
concentration,the
effect of a competitive
inhibitor can be
overcome,
Succinate dehydrogenase
Reversible Competitive inhibition
On a Lineweaver-Buck plot a competitive can
be seen to increase the Km but leave Vmax
unchanged
Reversible competitive inhibition
Reversible noncompetitive
inhibition
? A noncompetitive
inhibitor binds at a site
other than the active
site of the enzyme and
decreases its catalytic
rate by causing a
conformational change
in the three-dimen-
sional shape of the
enzyme,
Reversible noncompetitive
inhibition
? The enzyme may bind
the inhibitor,the
substrate or both the
inhibitor and substrate
together,
Reversible noncompetitive
inhibition
? The effect of a noncompetitive inhibitor
cannot be overcome at high substrate
concentrations,
? On a Lineweaver-Buck plot a noncompetitive
inhibitor can be seen to decrease the Vmax
but leave Km unchanged,
Lineweaver-Buck plot
Competitive inhibition and
noncompetitive inhibition
Competitive inhibition and
noncompetitive inhibition
C5 regulation of enzyme activity
1,Feedback regulation反馈调控
2,Allosteric enzyme别构酶
3,Reversible covalent modification
4,Proteolytic activation蛋白水解的活化
5,Regulation of enzyme synthesis and
breakdown
Feedback regulation反馈调控
? The rate of enzyme-catalyzed reactions in biological
systems are altered by activators and inhibitors,
(collectively known as effectors.)
In metabolic pathways,the end-product often feedback-
inhibits (when an enzyme early on in the pathway is
inhibited by an end-product of the metabolic
pathway)the committed step earlier in the same
pathway to prevent the build up of intermediates and
the unnecessary use of metabolites and energy,
Feedback inhition loop
Sequential feedback inhibition
For branched metabolic pathways a process of
sequential feedback inhibition often operates,
Allosteric enzyme
? A plot of V 0 against [S] for an allosteric enzyme
gives a sigmoidal-shaped curve,
Allosteric enzyme
1,Allosteric enzymes are often multi-subunit
proteins,with an active site on each subunit,
2,They often have more than one active site which
co-operatively bind substrate molecules,such that
the binding of substrate at one active site induces a
conformational change in the enzyme that alters the
affinity of the other active sites for substrates,
3,In addition,allosteric enzymes may be controlled by
effectors (activators or inhibitors) that bind to a site
other than the active site and alter the rate of
enzyme activity,
Allosteric enzyme
Aspartate transcarbamonylase
ATCase 天冬氨酸转氨甲酰酶
? This enzyme consist of six catalytic subunits each
with an active site and six regulatory subunits to
which the allosteric effectors cytosine triphosphate
(CTP) and ATP bind,
Allosteric activator
? Aspartate transcarbamoylase is feedback-inhibitor,
? In contrast,ATP an intermediate earlier in the
pahway,acts as an allosteric activator,
Allosteric
Reversible covalent modification
? The activity of many enzymes is alterd by the
reversible making and breaking of a covalent bond
between the enzyme and a small nonprotein group,
Covalent modifation
Phosphorylation and
dephosphorylation
? The most common such modification is the addition
and removal of a phosphate group;phosphorylation
and dephosphorylation去磷酸化,respectively,
Phosphorylation is catalyzed by protein kinases,蛋白
激酶 often using ATP as the phosphate donor,
whereas dephosphorylation is catalyzed by protein
phosphatases蛋白磷酸化酶。
Phosphorylation
Phosphorylation and
dephosphorylation
Proteolytic activation
? Some enzymes are synthesized as larger inactive
precursors calles proenzymes or zymogens酶原,
? These are activated by the irreversible hydrolysis of
one or more pepide bonds,
? The pancreatic proteases trypsin,chymotrypsin and
elastase are all derived from zymogen precursors by
proteolytic activation,
? Premature activation of these zymogens leads to the
condition of acute pancreatitis急性胰腺炎,
The central role of trypsin
Activation of chymotrypsinogen
The blood clotting cascade
? The blood clotting cascade also involves a series of
zymogen activations that brings about a large
amplification of the original signal,
Clotting cascade
Clotting cascade
Regulation of enzyme synthesis
and breakdown
? The amount of enzyme present is a balance between
the rates of its synthesis and degradation,The level
of induction or repression of the gene encoding the
enzyme,and the rate of degradation of its mRNA,will
alter the rate of synthesis of the enzyme protein,
? Once the enzyme protein has been synthesized,the
rate of its breakdown (half-life) can also be altered a
means of regulating enzyme activity,
cAMP
cAMP
Example one
Chymotrypsin
? (a) A representation of primary
structure,showing disulfide bonds
and the location of key amino acids,
Note that the protein consists of
three polypeptide chains,The active-
site amino acids are found grouped
together in the three-dimensional
structure,
? (b) A space-filling model of
chymotrypsin,The pocket in which
the aromatic amino acid side chain is
bound is shown in green,
? (c) The polypeptide backbone of
chymotrypsin shown as a ribbon
structure,Disulfide bonds are shown
in yellow; the A,B,and C chains are
shown in dark blue,light blue,and
white,respectively,
Chymotrypsin
? (d) A close-up of the chymotrypsin active site with a substrate bound,
Ser195 attacks the carbonyl group
of the substrate (shown in purple);
the developing negative charge on
the oxygen is stabilized by the
oxyanion hole (amide nitrogens
shown in orange) In the substrate,
the aromatic amino acid side
chain and the amide nitrogen of
the peptide bond to be cleaved
are shown in light blue,
Chymotrypsin
? C2 Thermodynamics热力学
? C3 Enzyme kinetics动力学
? C4 Enzyme inhibition
? C5 Regulation of enzyme
activity
C Enzymes
? Enzyme as catalysts催化剂
? Active site 活性部位
? Substrate specificity底物专一性
? Enzyme classification分类
? Enzyme assays酶的测定
? Linked enzyme assays
? Coenzymes辅酶 and prosthetic groups辅基
? Isoenzymes同工酶
C1 Introduction to enzymes
Enzymes as catalysts
? Enzymes are catalysts that change the rate of a
reaction without being changed themselves
? Enzyme catalyzed reactions usually take place under
relatively mild conditions(T,P,pH),
? Enzymes are highly specific and their activity can be
regulated,
? Virtually all enzymes are proteins,although some
catalytically active RNAs have been identified,
Active site活性部位
? Active site is the region that binds the substrate 底物
and converts it into product。
? 结合部位：负责与底物的结合，决定酶的专一性。
? 催化部位：负责催化底物键的断裂形成新键，决定酶的催
化能力。
? Small（ 1-2%）,a three-dimensional entity实体
? Cleft or crevice裂缝 on the surface of enzyme
? Bind by multiple weak forces
? Enzyme-substrate complex
? transition state complex过渡态复合物 --?product
Active site
Active site
Enzyme-substrate complex
Two models---enzyme bind its
substrate
? Lock and key model锁 -钥,Emil Fischer,1894
? Induced fit model诱导契合,Daniel E,
Koshland,Jr 1958
Lock and key model
Induced fit model
Induced fit model
? Active sites in the uninduced enzyme are shown schematically with
round contours,Binding of the first substrate (gold) induces a
conformational shift (angular contours) that facilitates binding of the
second substrate (blue),with far lower energy than otherwise
required,When catalysis is complete,the product is released,and
the enzyme returns to its uninduced state,
Substrate specificity
? Substrate specificity is often determined by
changes in relatively few amino acids in the
active site,
? 结构专一性,
1) 绝对专一性（脲酶）；
2) 相对专一性（酶作用于一类结构相近的底物）
? 立体异构专一性
1) 旋光异构专一性（ L-氨基酸氧化酶）
2) 几何异构专一性
Serine proteases丝氨酸蛋白酶家族
? Trypsin胰蛋白酶 cleaves on the carboxyl side
of positively charged Lys or Arg residues,
? Chymotrypsin 胰凝乳蛋白酶, cleaves on the
carboxyl side of bulky aromatic and
hydrophobic amino acid residues
? Elastase 弹性蛋白酶, cleaves on the carboxyl
side of small uncharged side chains
COO
-
C
A s p
COO
-
C
A s p
T r y p sin Ch y m o tr y p sin E last a se
切 L y s,Ar g 切 T rp,Phe,T y r 切 Ala,Gl y
Non - p o l a r
p o ck e t
Dee
p
a
n
d
n
e
g
a
tiv
e
l
y
ch
a
r
g
e
d
p
o
ck
e
t
S h a l l o w a n d
n o n - p o l a r
p o ck e t
O O
– C – N – C – C – N –
C
C
C
C
NH
3
+
O O
– C – N – C – C – N –
C
O O
– C – N – C – C – N –
CH
3
Ju
a
n
g
R
H
(2
0
0
4
)
BC
b
a
si
cs
Enzyme classification
2,Enzyme classification
? 氧化 -还原酶催化氧化 -还原反应。
? 主要包括脱氢酶 (dehydrogenase)和氧化酶 (Oxidase)。
? 如，乳酸 (Lactate)脱氢酶催化乳酸的脱氢反应。
(1) 氧化 -还原酶 Oxidoreductase
C H 3 C H C O O H
O H
N A D + H +C H 3 C C O O H
O
N A D H
2,Enzyme classification
? 转移酶催化基团转移反应，即将一个底物分子的基团或原子转移到
另一个底物的分子上。
例如,谷丙转氨酶催化的氨基转移反应。
(2) 转移酶 Transferase
C H 3 C H C O O H
N H 2
H O O C C H 2 C H 2 C C O O H
O
H O O C C H 2 C H 2 C H C O O H
N H 2
C H 3 C C O O H
O
2,Enzyme classification
? 水解酶催化底物的加水分解反应。
? 主要包括淀粉酶、蛋白酶、核酸酶及脂酶等。
? 例如，脂肪酶 (Lipase)催化的脂的水解反应,
(3) 水解酶 hydrolase
H 2 OC O O C H 2 C H 3R R C O O H C H 3 C H 2 O H
Enzyme classification
? 裂合酶催化从底物分子中移去一个基团或原子形成双键
的反应及其逆反应。
? 主要包括醛缩酶、水化酶及脱氨酶等。
? 例如,延胡索酸水合酶催化的反应。
(4) 裂合酶 Lyase
H O O C C H = C H C O O H H 2 O H O O C C H 2 C H C O O H
O H
Enzyme classification
? 异构酶催化各种同分异构体的相互转化，即底物分子内
基团或原子的重排过程。
例如,6-磷酸葡萄糖异构酶催化的反应。
(5) 异构酶 Isomerase
O
C H 2 O H
O H
O H
O H
O H O
C H 2 O H C H 2 O H
O H
O H
O H
Enzyme classification
? 合成酶，又称为连接酶，能够催化 C-C,C-O,C-N 以及
C-S 键的形成反应。这类反应必须与 ATP分解反应相互
偶联。
? A + B + ATP + H-O-H ===A ? B + ADP +Pi
? 例如，丙酮酸羧化酶催化的反应。
丙酮酸 + CO2 ? 草酰乙酸
(6) 合成酶 Ligase or Synthetase
Enzyme classification
? 核酸酶是唯一的非蛋白酶。它是一类特殊的 RNA，能够催化 RNA分子
中的磷酸酯键的水解及其逆反应。
(7) 核酸酶（催化核酸） ribozyme
3'3'
3'3'3'
3'3'3'3'
3'
5'5'5'5'
5'
5'5'5'5'5'
BBBBB
PP P
+
P O HPP
BBBB
PPPP
P
B
P
Enzyme classification
? Trypsin’s Enzyme Commission number is 3.4.21.4
? 3 denote it is a hydrolase
? 4 denote it is a protease that hydrolyzes peptide
bonds
? 21 denote it is a serine protease with a serine
residue at the active site
? 4 indicates that it was the fourth enzyme to be
assigned to this class,
Enzyme assays
? The amount of enzyme protein present can be
determined in terms of the catalytic effect it
produces,that is the conversion of substrate
to product,
1,The overall equation of the reaction
2,The disappearance of substrate or the
appearance of product
3,Cofactors,pH,temperature
4,Sufficient supply of substrate
Enzyme assay
? Enzyme activity（酶催化某一化学反应的能
力）,the rate of appearance of product or the
rate of disappearance of substrate
? Absorbance:spectrophotometer分光光度计
? Two most common molecules,
? NADH(reduced nicotinamide ademine
dinucleotied还原型烟酰胺腺嘌呤二核苷酸 )
? NADPH:340nm
NADH (NADPH)
LDH:Lactate dehydrogenase
Linked enzyme assays
? If neither the substrates nor products of
an enzyme-catalyzed reaction absorb light
at an appropriate wavelength,the enzyme
can be assayed by linking to another
enzyme-catalyzed reaction that does
involve a change in absorbance,
? The second enzyme must be in excess,so
that the rate-limit step in the linked assay
is the action of the first enzyme,
Coenzymes and prosthetic groups
? Cofactors:small,nonprotein units(iorganic ions or a
complex organic molecule called coenzyme辅酶 )
? Prosthetic group 辅基,A metal or coenzyme that is
covalently attached to the enzyme is called a
prosthetic group,
? Holoenzyme全酶 =cofactor辅因子 +apoenzyme脱辅酶
? Many coenzymes are derived from vitamin
precursors,giving rise to deficiency diseases when
in inadequate supply,
? P73 table 2
Cofactors
NAD+（辅酶 I） and NADP+(辅酶 II)
? NAD+ and NADP+ have a common function as they
both act as carriers of electrons and are involved in
oxydation-reduction reactions,
? NAD+ is more commonly used in catabolic(break
down)分解代谢 reactions
? NADP+ is used in anabolic(biosynthetic)合成代谢
reactions,
? The reactive part of both molecules is the
nicotinamide ring which exists in reduced or an
oxidized form,and so acts to accept or donate
electrons in an enzyme reaction,
? NAD+ +H++2e- < ----- > NADH
NAD+
NADP
FAD and FMN
? Flavin adenine dinucleotide(FAD) 黄素腺嘌呤二核 苷
酸 and flavin mononucleotide(FMN) 黄素单核苷酸 are
also carriers of electrons,
? Reactive site is flavine mononucleotide unit,
? FAD and FMN react with two protons as well as two
electrons,in alternating between the reduced and
oxidized state,
? FAD+2H++2e-< ----- >FADH2
FMN
FAD
Isoenzymes:同工酶
? Isoenzymes are different forms of an
enzyme which catalyze the same
reaction,but which exhibit different physical
or kinetic properties(pI,pH,substrate
affinity or inhibitors)
? LDH:乳酸脱氢酶
? CH3CH(OH)COO-+NAD+< ---->CH3COCOO-
+NADH+H+
LDH
? LDH is a tetramer of two different types of
subunits,called H and M,which have small
differences in amino acid sequence,
? The two subunits can combine randomly with each
other,forming 5 isoenzymes that have the
compositions H4,H3M,H2M2,HM3,M4,
? They can be resolved electrophoretically,
? M subnunits predominate in skeletal muscle and
liver,whereas H subunits predominate in the heart,
? H4,H3M:heart and red blood cells
? H2M2:brain and kidney
? HM3,M4,liver and skeletal muscle
C2 Thermodynamics
1,Thermodynamics热力学
2,Activation energy 活化能 and transition
state
3,Free energy change
4,Chemical equilibria化学平衡
Thermodynamics
? A knowledge of thermodynamics,(which is the
description among the various forms of energy and
how energy affects matter),enables one to determine
whether a physical process is possible,
? In Thermodynamics,a system is the matter within a
defined region,
? The matter in the rest of the universe is called the
surroundings,
The first laws of thermodynamics
? ΔE=EA-EB=Q-W
? EA is the energy of the system at the start of a
process
? EB is the energy of the system at the end of the
process
? Q is the heat absorbed by the system
? W is the work done by the system,
? Q = ΔE+ W 系统从外界吸收的热量等于系统内能
的增量和系统对外界做功之和 。这就是热力学第
一定律。
The second laws of thermodynamics
? The second laws of thermodynamics states that a
process can occur spontaneously only if the sum of
the entropies 熵 (Entropy is a measure of the degree
of randomness or disorder of a system)of the
system and its surroundings increases,
? ΔS system+ Δ S surroundings >0 for a spontaneous
process
? Entropy changes of chemical reactions are not
readily measured,
Free energy自由能 (G)
? Josiah Willard Gibbs
? The first and second laws of thermodynamics are
combined in the thermodynamic function,free
energy,
? ΔG= ΔH-T ΔS(ΔH:热量的变化； ΔS：熵变 )
? ΔH= ΔE+P ΔV
? ΔG= ΔE-T ΔS
? If △ G is negative,that reaction can happen
spontaneously
? If △ G is positive,an input of energy is required to
drive the reaction,
Activation energy and transition state
For a biochemical reaction to proceed,the
energy barrier needed to transform the
substrate molecules into the transition state
has to overcome,
The transition state has the highest free energy in
the energy pathway,
Gibbs 活化自由能
ΔG++（ Gibbs 活化自由能 ）,
The difference in free
energy between the
substrate and
transition state is
termed the Gibbs free
energy of activation,
△ G ++
An enzyme stabilizes the
transition state and
lowers △ G ++,thus
increasing the rate at
which the reaction
occurs,
△ G ++ and △ G
△ Go’
? ATP+H2O------->ADP+Pi
? △ Go’=-30.5kJ mol-1=-7.3kcalmol-1
? 1kcal卡 =4.184kJ
Chemical equilibria
? A chemical reaction often exists in a state of dynamic
equilibrium,
? The equilibrium constant ( K) defines the ratio of the substrates
and product at equilibrium,
? Enzymes do not alter the equilibrium position,but do accelerate
the attainment of the equilibrium position by speeding up the
forward and reverse reactions,
C3 Enzyme kinetics
? Enzyme velocity
? Substrate Concentration
? Enzyme concentration
? Temperature
? pH
? Michaelis-menten Model
? Lineweaver-Burk Plot
Enzyme velocity
? Enzyme activity is commonly expressed by the
intial rate ( V0) of the reaction being catalyzed,
The units of V 0are u mol/ min。 (why?)
Enzyme unit
? Enzyme activity may be expressed in a number of
ways,the commonest is by the V0
? There are also 2 standard units of enzyme activity,
? the enzyme unit (U) and the katal(kat),
? 1U,the amount of enzyme which will catalyze the
transformation of 1 u mol of substrate per min at
25oC under optimal conditions,
? 1kat:在最适条件下，每秒钟能催化 1mol底物转化为产物
所需的酶量。
? 1U=16.67 nkat,
Specific activity比活力
The specific activity is the number of units per
milligram of protein(units /mg)
Or 每 g酶制剂或每 ml酶制剂含有多少个活力单位表
示（ U/g,U/ml）。用来比较每单位质量蛋白质的
催化能力。
? The specific activity is a measure of the
purity of an enzyme,
Substrate Concentration
? At low substrate concentrations,a doubling of [S]
leads to a doubling of V 。,whereas at higher [S] the
enzyme becomes saturated and there is no further
increase in V 。
Substrate Concentration
1,中间复合物学说
2,饱和现象
3,双曲线
Enzyme concentration
? When [S] is saturating,a doubling of the
enzyme concentration leads to a
doubling of V0,
Temperature
? Temperature affects the rate of an enzyme-catalyzed
reaction by increasing the thermal energy of the
substrate molecules,
? This increases the proportion of molecules with
sufficient energy to overcome the activation barrier
and hence increases the rate of the reaction,
? in addition,the thermal energy of the component
molecules of the enzyme is increased,which leads to
an increased rate of denaturation of the enzyme
protein due to the disruption of the noncovalent
interactions holding the structure together,
T
pH
? Each enzyme has an optimum pH at which the rate of
the reaction that it catalyzes is at its maximum,
? Slight deviations in the pH from the optimum lead to
a decrease in the reaction rate,
? Larger deviations in pH lead to denaturation of the
enzyme due to charges in the ionization of amino
acid residues and the disruption of noncovalent
interactions
pH
Michaelis-menten Model
Vmax指该酶促反应的最大速度,[S]为底物浓度,Km是米氏常数,VO是在
某一底物浓度时相应的反应速度。
Michaelis-Menten model
Michaelis-Menten model
Michaelis-Menten model
[E]+[S]------->[ES] k1
[ES]-----> [E]+[S] K-1
[ES]------- >[E]+[P] k2
Michaelis-Menten model
Michaelis-Menten model
? The concentration of uncombined enzyme [E] is equal
? to the total enzyme concentration [E]T minus the
concentration of the [ES]complex,
Michaelis-Menten model
Michaelis-Menten model
? The maximal rate,Vmax,is attained when the catalytic
sites on the enzyme are saturated with substrate— that is,
when [ES] = [E]T,Thus,
A direct plot
当反应速度等于
最大速度一半时,
即 V = 1/2 Vmax,
Km = [S]
Km
Kcat:催化常数，转换数,每秒钟每个酶
分子转换底物的分子数。
A lineweaver-burk double-
reciprocal plot
C4 Enzyme inhibition
1,Enzyme inhibition
2,Irreversible inhibition
3,Reversible competitive inhibition
4,Reversible noncompetitive inhibition
Enzyme inhibition
? Inhibitor:any molecule which acts directly
on an enzyme to lower its catalytic rate is
called an inhibitor.(not denaturation)
? Some enzyme inhibitors are normal body
metabolites,
? other may be foreign substances,such as
drugs or toxins
Irreversible
? Irreversible:An irreversible inhibitor binds
tightly,often covalently,to amino acid
residues at the active site of the enzyme,
permanently inactivating the enzyme,
1.非专一性不可逆抑制
2.专一性不可逆抑制
diisopropylfluorophosphate( DIPF)
Iodoacetamide 碘乙酰胺
Antibiotic penicillin
Penicillin
Irreversible
e.g
Reversible Competitive inhibition
A competitive inhibitor
typically has colse
structural similarities to
the normal substrate for
the enzyme,
Thus it competes with the
substrate molecules for
binding to the active
site of the enzyme,
Reversible Competitive inhibition
? At high substrate
concentration,the
effect of a competitive
inhibitor can be
overcome,
Succinate dehydrogenase
Reversible Competitive inhibition
On a Lineweaver-Buck plot a competitive can
be seen to increase the Km but leave Vmax
unchanged
Reversible competitive inhibition
Reversible noncompetitive
inhibition
? A noncompetitive
inhibitor binds at a site
other than the active
site of the enzyme and
decreases its catalytic
rate by causing a
conformational change
in the three-dimen-
sional shape of the
enzyme,
Reversible noncompetitive
inhibition
? The enzyme may bind
the inhibitor,the
substrate or both the
inhibitor and substrate
together,
Reversible noncompetitive
inhibition
? The effect of a noncompetitive inhibitor
cannot be overcome at high substrate
concentrations,
? On a Lineweaver-Buck plot a noncompetitive
inhibitor can be seen to decrease the Vmax
but leave Km unchanged,
Lineweaver-Buck plot
Competitive inhibition and
noncompetitive inhibition
Competitive inhibition and
noncompetitive inhibition
C5 regulation of enzyme activity
1,Feedback regulation反馈调控
2,Allosteric enzyme别构酶
3,Reversible covalent modification
4,Proteolytic activation蛋白水解的活化
5,Regulation of enzyme synthesis and
breakdown
Feedback regulation反馈调控
? The rate of enzyme-catalyzed reactions in biological
systems are altered by activators and inhibitors,
(collectively known as effectors.)
In metabolic pathways,the end-product often feedback-
inhibits (when an enzyme early on in the pathway is
inhibited by an end-product of the metabolic
pathway)the committed step earlier in the same
pathway to prevent the build up of intermediates and
the unnecessary use of metabolites and energy,
Feedback inhition loop
Sequential feedback inhibition
For branched metabolic pathways a process of
sequential feedback inhibition often operates,
Allosteric enzyme
? A plot of V 0 against [S] for an allosteric enzyme
gives a sigmoidal-shaped curve,
Allosteric enzyme
1,Allosteric enzymes are often multi-subunit
proteins,with an active site on each subunit,
2,They often have more than one active site which
co-operatively bind substrate molecules,such that
the binding of substrate at one active site induces a
conformational change in the enzyme that alters the
affinity of the other active sites for substrates,
3,In addition,allosteric enzymes may be controlled by
effectors (activators or inhibitors) that bind to a site
other than the active site and alter the rate of
enzyme activity,
Allosteric enzyme
Aspartate transcarbamonylase
ATCase 天冬氨酸转氨甲酰酶
? This enzyme consist of six catalytic subunits each
with an active site and six regulatory subunits to
which the allosteric effectors cytosine triphosphate
(CTP) and ATP bind,
Allosteric activator
? Aspartate transcarbamoylase is feedback-inhibitor,
? In contrast,ATP an intermediate earlier in the
pahway,acts as an allosteric activator,
Allosteric
Reversible covalent modification
? The activity of many enzymes is alterd by the
reversible making and breaking of a covalent bond
between the enzyme and a small nonprotein group,
Covalent modifation
Phosphorylation and
dephosphorylation
? The most common such modification is the addition
and removal of a phosphate group;phosphorylation
and dephosphorylation去磷酸化,respectively,
Phosphorylation is catalyzed by protein kinases,蛋白
激酶 often using ATP as the phosphate donor,
whereas dephosphorylation is catalyzed by protein
phosphatases蛋白磷酸化酶。
Phosphorylation
Phosphorylation and
dephosphorylation
Proteolytic activation
? Some enzymes are synthesized as larger inactive
precursors calles proenzymes or zymogens酶原,
? These are activated by the irreversible hydrolysis of
one or more pepide bonds,
? The pancreatic proteases trypsin,chymotrypsin and
elastase are all derived from zymogen precursors by
proteolytic activation,
? Premature activation of these zymogens leads to the
condition of acute pancreatitis急性胰腺炎,
The central role of trypsin
Activation of chymotrypsinogen
The blood clotting cascade
? The blood clotting cascade also involves a series of
zymogen activations that brings about a large
amplification of the original signal,
Clotting cascade
Clotting cascade
Regulation of enzyme synthesis
and breakdown
? The amount of enzyme present is a balance between
the rates of its synthesis and degradation,The level
of induction or repression of the gene encoding the
enzyme,and the rate of degradation of its mRNA,will
alter the rate of synthesis of the enzyme protein,
? Once the enzyme protein has been synthesized,the
rate of its breakdown (half-life) can also be altered a
means of regulating enzyme activity,
cAMP
cAMP
Example one
Chymotrypsin
? (a) A representation of primary
structure,showing disulfide bonds
and the location of key amino acids,
Note that the protein consists of
three polypeptide chains,The active-
site amino acids are found grouped
together in the three-dimensional
structure,
? (b) A space-filling model of
chymotrypsin,The pocket in which
the aromatic amino acid side chain is
bound is shown in green,
? (c) The polypeptide backbone of
chymotrypsin shown as a ribbon
structure,Disulfide bonds are shown
in yellow; the A,B,and C chains are
shown in dark blue,light blue,and
white,respectively,
Chymotrypsin
? (d) A close-up of the chymotrypsin active site with a substrate bound,
Ser195 attacks the carbonyl group
of the substrate (shown in purple);
the developing negative charge on
the oxygen is stabilized by the
oxyanion hole (amide nitrogens
shown in orange) In the substrate,
the aromatic amino acid side
chain and the amide nitrogen of
the peptide bond to be cleaved
are shown in light blue,
Chymotrypsin
