第 12章 氨基酸代谢
第一节 The nitrogen cycle
Nitrogen exists predominantly in an oxidized state
in the environment,occurring principally as N2 in the
atmosphere or as nitrate ion (NO3-) in the soils and
oceans,Its acquisition by biological systems is
accompanied by its reduction to ammonium ion
(NH4+) and the incorporation of NH4+ into organic
linkage as amino group,The reduction of NO3- to
NH4+ occurs in green plants,various fungi,and
certain bacteria in a two-step metabolic pathway
known as nitrate assimilation.
第一节 The nitrogen cycle
The formation of NH4+ from N2 gas is termed
nitrogen fixation,N2 fixation is an exclusively
prokaryotic process,No animals are capable of either
nitrogen fixation or nitrate assimilation.
Animals release excess nitrogen in a reduced form,
either as NH4+ or as organic nitrogenous compounds
such as urea,The release of N occurs both during life
and as a consequence of microbial decomposition
following death.
第一节 The nitrogen cycle
Various bacteria return the reduced forms of
nitrogen back to the environment by oxidizing them.
The oxidation of NH4+ to NO3- is performed by
nitrifying bacteria,Nitrate nitrogen also returns to
the atmosphere as N2 as result of the metabolic
activity of denitrifying bacteria.
第一节 The nitrogen cycle
Dietary proteins are digested into
amino acids in the gastrointestinal(胃
肠 ) tract via the action of pepsin,
trypsin,chymotrypsin,
carboxypeptidases and
aminopeptidases.
Sources of amino acids for animals
Proteins (but
not pepsin) unfolded
Absorbed as tri- & dipeptides,
and amino acidsDegradation & absorption
of dietary proteins
Pepsin,the first enzyme
discovered (18th century).
proteases
Essential amino acids
Amino acids can not be stored in
animals,excess being completely
oxidized to release energy or
converted to storable fuels (fatty
acids or carbohydrates).
Overall fate of excess amino acids
第二节 Amino acid degradation
1,氧化脱氨基
氨基酸在酶的作用下脱去氨基生成相应酮酸的过
程，叫氧化脱氨基作用。
一, 氨的去路
RCHCOO ——
-|
NH
3
+
R C C O O——
-
NH
3
+
|
R C C O O + H——
- +
|
O
|
FA D
F M N（）
FA D H
F M N H
2
（）
2
HO
2
NH
3
HO
2 2
O
2
氨基酸氧化酶
Glu + NAD(P) + H2O? a-KG + NH4+ + NADH(P) + H+
2,脱氢酶作用 -GDH
一, 氨的去路
NH
3
+
|
H C CO O——
-
|
CH
2
|
CH
2
|
CO O
-
+ N A D P（）
+
+ HO
2
N H +
4
+
+ N A D P H （） + H
+
O
||
C CO O—
-
|
CH
2
|
CH
2
|
CO O
-
L - 谷氨酸 - 酮戊二酸 α
谷氨酸脱氢酶
3,转氨基作用 一, 氨的去路
R1
|
H C NH—— 2
|
COOH
R2
|
C=O
|
COOH
+
R1
|
H C NH—— 2
|
COOH
R2
|
C=O
|
COOH
+
转氨酶
转氨基作用是 α-氨基酸和 α-酮酸之间氨基的转移作用
。一种 α-氨基酸的 α-氨基借助转氨酶（ transaminase） 的催
化作用转移到 α-酮酸的羰基上，结果生成新的酮酸，而原
来的 α-酮酸则形成相应的氨基酸。
3,转氨基作用
谷丙转氨酶催化的转氨基作用机理
一, 氨的去路
CH
3
|
H C N H——
2
|
CO O H
HO —
HC
3
—
H C = O
|
O
||
|
O
-
— ——— C H O P O
2
-
CH
3
|
C = O
|
CO O H N
HO —
HC
3
—
C H N H
2
—
2
|
O
||
|
O
-
— ——— C H O P O
2
-
（） CH
22
CO O H
|
|
C H N H—
2
|
CO O H
（） CH
22
CO O H
|
|
C = O
|
CO O H
丙氨酸 磷酸吡哆醛 谷氨酸
丙酮酸 磷酸吡哆胺 α - 酮戊二酸
4,联合脱氨作用 (转氨酶 -谷氨酸脱氢酶 )
谷氨酸
α-酮戊二酸丙 氨酸
丙酮酸
转氨酶 谷氨酸脱氢酶
NAD(P)+H+
NAD(P)+
联合脱氨基作用
PLP
一, 氨的去路
在氨基酸脱羧酶催化下进行脱羧作用，生成一个
伯胺类化合物和 CO2,其反应可以用下式表示
二, 脱羧基作用
NH 2
|
R CH COOH —— R CH— 2
NH 2
|氨基酸脱羧酶
PLP
+ CO 2
PLP acts as a temporary
carrier of amino groups
at the active sites of
all aminotransferases.
PLP facilitates several
different types of
transformation around
the a-carbon of
amino acids.
PLP is derived from
vitamin B6 (pyridoxine，
吡哆醇 )
吡哆醛磷酸
磷酸吡哆胺
Serum aminotransferases have been
used as clinical markers of tissue
damages
? Damaged heart or liver cells leak
aminotransferases.
? Blood aspartate aminotransferase and
alanine aminotransferase are usually
examined for indications of illness.
三, 氨基酸碳架的分解
氨基酸脱羧酶
1,进入 TCA循环
Oxidation of the
carbon skeletons of
amino acids in
mammals
2.再合成为氨基酸
谷氨酸＋丙酮酸 α-酮戊二酸＋丙氨酸
谷氨酸＋草酰乙酸 α-酮戊二酸＋天冬氨酸
三, 氨基酸碳架的分解
NH 3
+
|
H C COO——
-
|
CH 2
|
CH
2
|
COO
-
+ NAD P（）
+
+ HO
2
NH +
4
+
+ NAD P H （） + H
+
O
||
C COO—
-
|
CH
2
|
CH
2
|
COO
-
3.转变为糖和脂肪
当体内不需要将 α -酮酸再合成氨基酸，并且体
内的能量供给充足时,α -酮酸可以转变为糖或脂肪
。例如，用氨基酸饲养患人工糖尿病的狗，大多数氨
基酸可使尿中的葡萄糖的含量增加，少数几种可使葡
萄糖及酮体的含量同时增加。 在体内可以转变为糖的
氨基酸称为 生糖氨基酸,按糖代谢途径进行代谢；能
转变为酮体的氨基酸称为 生酮氨基酸。
三, 氨基酸碳架的分解
硝酸盐还原分两步进行：第一步在硝酸还原酶
（ nitrate reductase,NR） 催化下, 由 NAD（ P） H提供 1对
电子, 硝酸盐被还原为亚硝酸盐, 第二步是在亚硝酸还原
酶 （ nitrite reductase,NiR） 下, 由还原型铁氧还蛋白
（ Fdred） 提供 3对电子, 使亚硝酸盐 （ NO2-） 还原成氨 。
第三节 Nitrate reduction
NR
NO
3
-
＋
2H ＋ 2e
-
NO
2
-
＋
H O
2
N iR
NO
2
-
＋
12H
+
＋
6e
-
NH
4
+
＋
2H O
2
硝酸盐还原分两步进行：第一步在硝酸还原酶
（ nitrate reductase,NR） 催化下, 由 NAD（ P） H提供
1对电子, 硝酸盐被还原为亚硝酸盐, 第二步是在亚硝
酸还原酶 （ nitrite reductase,NiR） 下, 由还原型铁氧
还蛋白 （ Fdred） 提供 3对电子, 使亚硝酸盐 （ NO2-）
还原成氨 。
第三节 Nitrate reduction
Ammonium enters organic linkage via three
major reactions that are found in all cells,The
enzymes mediating these reactions are：
(1) Cabamoyl-phosphate synthetase I (氨甲酰磷
酸合成酶 )
(2) Glutamate dehydrogenase（ 谷氨酸脱氢酶）,
(3) Glutamine synthetase（ 谷氨酰氨合成酶）,
第四节 Ammonium assimilation
NH4+ in hepatocytes (肝细胞 ) is convert ed
into urea for excretion via the urea cycle in
most terrestrial vertebrates
? Urea is formed from ammonia,CO2 (as
bicarbonate) and Asp.
? The pathway was also discovered by Hans
Krebs in 1932 (five years before he discovered
the citric acid cycle).
? Four ATP molecules are consumed to produce
each urea.
Carbamoyl-phosphate synthetase I catalyzes
one of the steps in the urea cycle,Two ATP are
consumed,one in the activation of HCO3- for
reaction with ammonium,and the other in the
phosphorylation of the carbamate formed:
1,Carbamoyl-phosphate synthetase I
NH4++HCO3-+2ATP?H2N-CO-O-PO3-+2ADP+Pi+2H+
N-acetylglutamate is an essential allosteric
activator for this enzyme
第四节 Ammonium assimilation
The synthesis of
Carbamoyl （ 氨甲酰）
phosphate requires
two activation
steps,consuming two
ATP molecules,one
for activating HCO3-,
the other to
phosphorylate
carbamate.
an anhydride
1,Carbamoyl-phosphate synthetase I
该反应消耗 2个 ATP分子中的两个高能磷酸键
,其中 1个是用于活化 HCO3-,另 1分子 ATP则用于
磷酸化氨甲酰基 。
第四节 Ammonium assimilation
Fumarate is converted back to
Asp via a partial usage of the
citric acid cycle.
The rate of urea synthesis is
controlled at two levels
? Allosteric （ 别构） regulation,N-acetylglutamate,by
binding to a site which hydrolyzes （ 水解） Gln in
another isozyme,positively regulates carbamoyl
phosphate synthetase I activity.
? Gene regulation,syntheses of the urea cycle enzymes
are all increased during starvation (when energy has to
be obtained from muscle proteins!) or after high protein
uptake.
? The rates of transcription of the five genes encoding the
enzymes are increased.
Genetic defects of the urea cycle
enzymes lead to hyperammonemia
and brain damage
? High levels of ammonia lead to mental disorder
or even coma and death.
? Ingenious strategies for coping with the
deficiencies have been devised based on a
thorough understanding of the underlying
biochemistry.
? Strategy I,diet control,provide the essential
amino acids in their a-keto acid forms.
? Strategy II,when argininosuccinate lyase is deficient,
ingesting a surplus of Arg will help (ammonia will be
carried out of the body in the form of argininosuccinate,
instead of urea).
? Strategy III,when carbamoyl phosphate synthetase I,
ornithine transcarbamoylase,or argininosuccinate
sythetase are deficient,the ammonia can be eliminated
by ingesting compounds (e.g.,benzoate or
phenylacetate),which will be excreted after accepting
ammonia.
Glutamate dehydrogenase catalyzes the reductive
amination of a-ketoglutarate to yield glutamate,Reduced
pyridine mucleotides (NADH or NADPH) provide the
reducing power:
2,Glutamate dehydrogenase (GDH)
NH4+ + a-ketoglutarate + NADPH+H+ ?
glutamate +NADP++H2O
第四节 Ammonium assimilation
The glutamate dehydrogenase reaction
第四节 Ammonium assimilation
3,Glutamine synthetase (GS)
Glutamine synthetase catalyses the ATP-dependent
amindation of the ?-carboxyl group of glutamate to form
glutamine,GS activity depends on the presence of
divalent cations such as Mg2+.
Glutamine is a major donor in the biosynthesis of
many organic N compounds and GS activity is tightly
regulated.
GDH and GS are responsible for most of the
ammonium assimilated into organic compounds.
第四节 Ammonium assimilation
谷氨酰胺合成酶
谷氨酰胺合成酶
第四节 Ammonium assimilation
The Glutamine synthetase is a
primary regulatory point in
nitrogen metabolism,being
regulated by at least eight
allosteric effectors and reversible
adenylylation.
The bacterial glutamine synthetase
has 12 subunits arranged as two
rings of hexamers.
Active
sites
Tyr397
(adenylylation site)
The glutamine
synthetase is
accumulatively
inhibited by at
least 8 allosteric
effectors,mostly
end products
of glutamine
metabolism.
Glutamate synthase catalyes the reductive amination of a-
ketoglutarate suing the amide-N of glutamine as the N
donor:
Glutamate synthase (GOGAT)
Reductant +a-KG+Gln ? 2 Glu+oxidized redctant
第四节 Ammonium assimilation
The glutamate synthase reaction
谷氨酸合酶
第四节 Ammonium assimilation
Only certain bacteria can fix N2 into ammonia
RhizobiaCyanobacteria
蓝细菌 根瘤菌
第 5节 Nitrogen fixation
The dinitrogenase (固氮酶 ) complex
in certain bacteria (diazotrophs)
catalyzes the conversion of N2
(azote,“without life”) to NH3,which
is the ultimate source of nitrogen for
all nitrogen-containing biomolecules.
N2 + 8H+ + 8e- 2NH3 + H2
The Haber method,N2 +3H2 2NH3 ?G`o = - 33.5kJ/mol
with iron catalyst,500oC,300 atmospheres.
The nitrogenase complex consists of dinitrogenase and dinitrogenase redutase
both being iron-sulfur proteins.
Dinitrogenase (a2b2)
or FeMo protein
Reductase,a dimer of two
Identical subunits bridged
by a 4Fe-4S,
ATP hydrolysis is coupled to
protein conformatinal changes.
Dinitrogenase
reductase (dimer)
or Fe protein
ADP
ADP
4Fe-4S
8Fe-7S
(P-cluster)
Fe-Mo cofactore
-
Fe-Mo cofactor
8Fe-7S
(P-cluster) 4Fe-4S
ADP
ADP
Molybdenum (or vanadium)
N2 is believed to
be reduced by the
Fe-Mo cofactorN
2
Fe
FeFe
Fe
FeFe
Fe
S
S
S
S
S
S
S
SS
Mo
高柠檬酸
Electrons are transferred through a
series of carriers to N2 for its
reduction on the nitrogenase
complex.
Electrons are
transferred
to N2 bound in
the active site
of dinitrogenase
via ferredoxin/
flavodoxin and
dinitrogenase
Reductase.
N2 + 8H+ +8e- + 16ATP + 16H2O
? 2NH3 + H2 + 16ADP + 16Pi
(or photophosphorylation)
Conformational change
reduces e- affinity
The oxidized dinitrogenase reductase
dissociates
from the dinitrogenase
Reduced dinitrogenase reductase
associates with the dinitrogenase
The nitrogenase complex is
extremely labile to O2 and various
protective mechanisms have
evolved,living anaerobically,
forming thick walls,uncoupling e-
transport from ATP synthesis
(entering O2 is used inmediately)or
being protected by O2-binding
proteins.
Genes encoding the protein
components of the nitrogenase
complex are being transferred
into non-nitrogen-fixing
bacteria and plants.
Reduced nitrogen in the form of
NH4+ is assimilated into amino
acids mainly via a two-enzyme
pathway, glutamine synthetase
and glutamate synthase (an
enzyme only present in bacteria
and plants).
Gln
synthetaseGlu
Synthase
(+NADPH
+ATP)
Gln
synthetase
The pathways for ammonia
to enter organic compounds.
Glu
Dehydrogenase
Very minor)
Asn
synthetase
Carbamoyl
Phosphate
Synthetase
Transamination
( or NH4+)
Summary
? Amino acid in excess can neither be stored,nor
excreted,but oxidized or converted.
? The amino groups and carbon skeletons of amino
acids take separate but interconnected pathways.
? Liver is the major site of amino acid degradation in
vertebrates.
? PLP facilitates the transamination and other
transformations of amino acids.
? Glutamate collects and delivers free ammonia to
the liver.
? Gln and Glu releases NH4+ in liver mitochondria.
? NH4+ in hepatocytes is converted into urea through
the urea cycle in most terrestrial vertebrates for
excretion.
? The conversion of ammonia to urea takes five (six)
enzymatic steps.
? The rate of urea synthesis is controlled at two
levels.
? The carbon skeletons of the amino acids are first
converted into seven major metabolic
intermediates.
? Some amino acids are converted to intermediates
of citric acid cycle by simple removal of the amino
groups.
? Acetyl-CoA is formed from the degradation of
many amino acids.
? O2 is used to break the aromatic rings of Pro,
Phe and Tyr,as well as to oxidize Cys.
? A few genetic diseases are related to defects of
Phe catabolism enzymes.
? Leu,Ile,and Val are degraded via reactions
similar to fatty acid oxidation.
第一节 The nitrogen cycle
Nitrogen exists predominantly in an oxidized state
in the environment,occurring principally as N2 in the
atmosphere or as nitrate ion (NO3-) in the soils and
oceans,Its acquisition by biological systems is
accompanied by its reduction to ammonium ion
(NH4+) and the incorporation of NH4+ into organic
linkage as amino group,The reduction of NO3- to
NH4+ occurs in green plants,various fungi,and
certain bacteria in a two-step metabolic pathway
known as nitrate assimilation.
第一节 The nitrogen cycle
The formation of NH4+ from N2 gas is termed
nitrogen fixation,N2 fixation is an exclusively
prokaryotic process,No animals are capable of either
nitrogen fixation or nitrate assimilation.
Animals release excess nitrogen in a reduced form,
either as NH4+ or as organic nitrogenous compounds
such as urea,The release of N occurs both during life
and as a consequence of microbial decomposition
following death.
第一节 The nitrogen cycle
Various bacteria return the reduced forms of
nitrogen back to the environment by oxidizing them.
The oxidation of NH4+ to NO3- is performed by
nitrifying bacteria,Nitrate nitrogen also returns to
the atmosphere as N2 as result of the metabolic
activity of denitrifying bacteria.
第一节 The nitrogen cycle
Dietary proteins are digested into
amino acids in the gastrointestinal(胃
肠 ) tract via the action of pepsin,
trypsin,chymotrypsin,
carboxypeptidases and
aminopeptidases.
Sources of amino acids for animals
Proteins (but
not pepsin) unfolded
Absorbed as tri- & dipeptides,
and amino acidsDegradation & absorption
of dietary proteins
Pepsin,the first enzyme
discovered (18th century).
proteases
Essential amino acids
Amino acids can not be stored in
animals,excess being completely
oxidized to release energy or
converted to storable fuels (fatty
acids or carbohydrates).
Overall fate of excess amino acids
第二节 Amino acid degradation
1,氧化脱氨基
氨基酸在酶的作用下脱去氨基生成相应酮酸的过
程，叫氧化脱氨基作用。
一, 氨的去路
RCHCOO ——
-|
NH
3
+
R C C O O——
-
NH
3
+
|
R C C O O + H——
- +
|
O
|
FA D
F M N（）
FA D H
F M N H
2
（）
2
HO
2
NH
3
HO
2 2
O
2
氨基酸氧化酶
Glu + NAD(P) + H2O? a-KG + NH4+ + NADH(P) + H+
2,脱氢酶作用 -GDH
一, 氨的去路
NH
3
+
|
H C CO O——
-
|
CH
2
|
CH
2
|
CO O
-
+ N A D P（）
+
+ HO
2
N H +
4
+
+ N A D P H （） + H
+
O
||
C CO O—
-
|
CH
2
|
CH
2
|
CO O
-
L - 谷氨酸 - 酮戊二酸 α
谷氨酸脱氢酶
3,转氨基作用 一, 氨的去路
R1
|
H C NH—— 2
|
COOH
R2
|
C=O
|
COOH
+
R1
|
H C NH—— 2
|
COOH
R2
|
C=O
|
COOH
+
转氨酶
转氨基作用是 α-氨基酸和 α-酮酸之间氨基的转移作用
。一种 α-氨基酸的 α-氨基借助转氨酶（ transaminase） 的催
化作用转移到 α-酮酸的羰基上，结果生成新的酮酸，而原
来的 α-酮酸则形成相应的氨基酸。
3,转氨基作用
谷丙转氨酶催化的转氨基作用机理
一, 氨的去路
CH
3
|
H C N H——
2
|
CO O H
HO —
HC
3
—
H C = O
|
O
||
|
O
-
— ——— C H O P O
2
-
CH
3
|
C = O
|
CO O H N
HO —
HC
3
—
C H N H
2
—
2
|
O
||
|
O
-
— ——— C H O P O
2
-
（） CH
22
CO O H
|
|
C H N H—
2
|
CO O H
（） CH
22
CO O H
|
|
C = O
|
CO O H
丙氨酸 磷酸吡哆醛 谷氨酸
丙酮酸 磷酸吡哆胺 α - 酮戊二酸
4,联合脱氨作用 (转氨酶 -谷氨酸脱氢酶 )
谷氨酸
α-酮戊二酸丙 氨酸
丙酮酸
转氨酶 谷氨酸脱氢酶
NAD(P)+H+
NAD(P)+
联合脱氨基作用
PLP
一, 氨的去路
在氨基酸脱羧酶催化下进行脱羧作用，生成一个
伯胺类化合物和 CO2,其反应可以用下式表示
二, 脱羧基作用
NH 2
|
R CH COOH —— R CH— 2
NH 2
|氨基酸脱羧酶
PLP
+ CO 2
PLP acts as a temporary
carrier of amino groups
at the active sites of
all aminotransferases.
PLP facilitates several
different types of
transformation around
the a-carbon of
amino acids.
PLP is derived from
vitamin B6 (pyridoxine，
吡哆醇 )
吡哆醛磷酸
磷酸吡哆胺
Serum aminotransferases have been
used as clinical markers of tissue
damages
? Damaged heart or liver cells leak
aminotransferases.
? Blood aspartate aminotransferase and
alanine aminotransferase are usually
examined for indications of illness.
三, 氨基酸碳架的分解
氨基酸脱羧酶
1,进入 TCA循环
Oxidation of the
carbon skeletons of
amino acids in
mammals
2.再合成为氨基酸
谷氨酸＋丙酮酸 α-酮戊二酸＋丙氨酸
谷氨酸＋草酰乙酸 α-酮戊二酸＋天冬氨酸
三, 氨基酸碳架的分解
NH 3
+
|
H C COO——
-
|
CH 2
|
CH
2
|
COO
-
+ NAD P（）
+
+ HO
2
NH +
4
+
+ NAD P H （） + H
+
O
||
C COO—
-
|
CH
2
|
CH
2
|
COO
-
3.转变为糖和脂肪
当体内不需要将 α -酮酸再合成氨基酸，并且体
内的能量供给充足时,α -酮酸可以转变为糖或脂肪
。例如，用氨基酸饲养患人工糖尿病的狗，大多数氨
基酸可使尿中的葡萄糖的含量增加，少数几种可使葡
萄糖及酮体的含量同时增加。 在体内可以转变为糖的
氨基酸称为 生糖氨基酸,按糖代谢途径进行代谢；能
转变为酮体的氨基酸称为 生酮氨基酸。
三, 氨基酸碳架的分解
硝酸盐还原分两步进行：第一步在硝酸还原酶
（ nitrate reductase,NR） 催化下, 由 NAD（ P） H提供 1对
电子, 硝酸盐被还原为亚硝酸盐, 第二步是在亚硝酸还原
酶 （ nitrite reductase,NiR） 下, 由还原型铁氧还蛋白
（ Fdred） 提供 3对电子, 使亚硝酸盐 （ NO2-） 还原成氨 。
第三节 Nitrate reduction
NR
NO
3
-
＋
2H ＋ 2e
-
NO
2
-
＋
H O
2
N iR
NO
2
-
＋
12H
+
＋
6e
-
NH
4
+
＋
2H O
2
硝酸盐还原分两步进行：第一步在硝酸还原酶
（ nitrate reductase,NR） 催化下, 由 NAD（ P） H提供
1对电子, 硝酸盐被还原为亚硝酸盐, 第二步是在亚硝
酸还原酶 （ nitrite reductase,NiR） 下, 由还原型铁氧
还蛋白 （ Fdred） 提供 3对电子, 使亚硝酸盐 （ NO2-）
还原成氨 。
第三节 Nitrate reduction
Ammonium enters organic linkage via three
major reactions that are found in all cells,The
enzymes mediating these reactions are：
(1) Cabamoyl-phosphate synthetase I (氨甲酰磷
酸合成酶 )
(2) Glutamate dehydrogenase（ 谷氨酸脱氢酶）,
(3) Glutamine synthetase（ 谷氨酰氨合成酶）,
第四节 Ammonium assimilation
NH4+ in hepatocytes (肝细胞 ) is convert ed
into urea for excretion via the urea cycle in
most terrestrial vertebrates
? Urea is formed from ammonia,CO2 (as
bicarbonate) and Asp.
? The pathway was also discovered by Hans
Krebs in 1932 (five years before he discovered
the citric acid cycle).
? Four ATP molecules are consumed to produce
each urea.
Carbamoyl-phosphate synthetase I catalyzes
one of the steps in the urea cycle,Two ATP are
consumed,one in the activation of HCO3- for
reaction with ammonium,and the other in the
phosphorylation of the carbamate formed:
1,Carbamoyl-phosphate synthetase I
NH4++HCO3-+2ATP?H2N-CO-O-PO3-+2ADP+Pi+2H+
N-acetylglutamate is an essential allosteric
activator for this enzyme
第四节 Ammonium assimilation
The synthesis of
Carbamoyl （ 氨甲酰）
phosphate requires
two activation
steps,consuming two
ATP molecules,one
for activating HCO3-,
the other to
phosphorylate
carbamate.
an anhydride
1,Carbamoyl-phosphate synthetase I
该反应消耗 2个 ATP分子中的两个高能磷酸键
,其中 1个是用于活化 HCO3-,另 1分子 ATP则用于
磷酸化氨甲酰基 。
第四节 Ammonium assimilation
Fumarate is converted back to
Asp via a partial usage of the
citric acid cycle.
The rate of urea synthesis is
controlled at two levels
? Allosteric （ 别构） regulation,N-acetylglutamate,by
binding to a site which hydrolyzes （ 水解） Gln in
another isozyme,positively regulates carbamoyl
phosphate synthetase I activity.
? Gene regulation,syntheses of the urea cycle enzymes
are all increased during starvation (when energy has to
be obtained from muscle proteins!) or after high protein
uptake.
? The rates of transcription of the five genes encoding the
enzymes are increased.
Genetic defects of the urea cycle
enzymes lead to hyperammonemia
and brain damage
? High levels of ammonia lead to mental disorder
or even coma and death.
? Ingenious strategies for coping with the
deficiencies have been devised based on a
thorough understanding of the underlying
biochemistry.
? Strategy I,diet control,provide the essential
amino acids in their a-keto acid forms.
? Strategy II,when argininosuccinate lyase is deficient,
ingesting a surplus of Arg will help (ammonia will be
carried out of the body in the form of argininosuccinate,
instead of urea).
? Strategy III,when carbamoyl phosphate synthetase I,
ornithine transcarbamoylase,or argininosuccinate
sythetase are deficient,the ammonia can be eliminated
by ingesting compounds (e.g.,benzoate or
phenylacetate),which will be excreted after accepting
ammonia.
Glutamate dehydrogenase catalyzes the reductive
amination of a-ketoglutarate to yield glutamate,Reduced
pyridine mucleotides (NADH or NADPH) provide the
reducing power:
2,Glutamate dehydrogenase (GDH)
NH4+ + a-ketoglutarate + NADPH+H+ ?
glutamate +NADP++H2O
第四节 Ammonium assimilation
The glutamate dehydrogenase reaction
第四节 Ammonium assimilation
3,Glutamine synthetase (GS)
Glutamine synthetase catalyses the ATP-dependent
amindation of the ?-carboxyl group of glutamate to form
glutamine,GS activity depends on the presence of
divalent cations such as Mg2+.
Glutamine is a major donor in the biosynthesis of
many organic N compounds and GS activity is tightly
regulated.
GDH and GS are responsible for most of the
ammonium assimilated into organic compounds.
第四节 Ammonium assimilation
谷氨酰胺合成酶
谷氨酰胺合成酶
第四节 Ammonium assimilation
The Glutamine synthetase is a
primary regulatory point in
nitrogen metabolism,being
regulated by at least eight
allosteric effectors and reversible
adenylylation.
The bacterial glutamine synthetase
has 12 subunits arranged as two
rings of hexamers.
Active
sites
Tyr397
(adenylylation site)
The glutamine
synthetase is
accumulatively
inhibited by at
least 8 allosteric
effectors,mostly
end products
of glutamine
metabolism.
Glutamate synthase catalyes the reductive amination of a-
ketoglutarate suing the amide-N of glutamine as the N
donor:
Glutamate synthase (GOGAT)
Reductant +a-KG+Gln ? 2 Glu+oxidized redctant
第四节 Ammonium assimilation
The glutamate synthase reaction
谷氨酸合酶
第四节 Ammonium assimilation
Only certain bacteria can fix N2 into ammonia
RhizobiaCyanobacteria
蓝细菌 根瘤菌
第 5节 Nitrogen fixation
The dinitrogenase (固氮酶 ) complex
in certain bacteria (diazotrophs)
catalyzes the conversion of N2
(azote,“without life”) to NH3,which
is the ultimate source of nitrogen for
all nitrogen-containing biomolecules.
N2 + 8H+ + 8e- 2NH3 + H2
The Haber method,N2 +3H2 2NH3 ?G`o = - 33.5kJ/mol
with iron catalyst,500oC,300 atmospheres.
The nitrogenase complex consists of dinitrogenase and dinitrogenase redutase
both being iron-sulfur proteins.
Dinitrogenase (a2b2)
or FeMo protein
Reductase,a dimer of two
Identical subunits bridged
by a 4Fe-4S,
ATP hydrolysis is coupled to
protein conformatinal changes.
Dinitrogenase
reductase (dimer)
or Fe protein
ADP
ADP
4Fe-4S
8Fe-7S
(P-cluster)
Fe-Mo cofactore
-
Fe-Mo cofactor
8Fe-7S
(P-cluster) 4Fe-4S
ADP
ADP
Molybdenum (or vanadium)
N2 is believed to
be reduced by the
Fe-Mo cofactorN
2
Fe
FeFe
Fe
FeFe
Fe
S
S
S
S
S
S
S
SS
Mo
高柠檬酸
Electrons are transferred through a
series of carriers to N2 for its
reduction on the nitrogenase
complex.
Electrons are
transferred
to N2 bound in
the active site
of dinitrogenase
via ferredoxin/
flavodoxin and
dinitrogenase
Reductase.
N2 + 8H+ +8e- + 16ATP + 16H2O
? 2NH3 + H2 + 16ADP + 16Pi
(or photophosphorylation)
Conformational change
reduces e- affinity
The oxidized dinitrogenase reductase
dissociates
from the dinitrogenase
Reduced dinitrogenase reductase
associates with the dinitrogenase
The nitrogenase complex is
extremely labile to O2 and various
protective mechanisms have
evolved,living anaerobically,
forming thick walls,uncoupling e-
transport from ATP synthesis
(entering O2 is used inmediately)or
being protected by O2-binding
proteins.
Genes encoding the protein
components of the nitrogenase
complex are being transferred
into non-nitrogen-fixing
bacteria and plants.
Reduced nitrogen in the form of
NH4+ is assimilated into amino
acids mainly via a two-enzyme
pathway, glutamine synthetase
and glutamate synthase (an
enzyme only present in bacteria
and plants).
Gln
synthetaseGlu
Synthase
(+NADPH
+ATP)
Gln
synthetase
The pathways for ammonia
to enter organic compounds.
Glu
Dehydrogenase
Very minor)
Asn
synthetase
Carbamoyl
Phosphate
Synthetase
Transamination
( or NH4+)
Summary
? Amino acid in excess can neither be stored,nor
excreted,but oxidized or converted.
? The amino groups and carbon skeletons of amino
acids take separate but interconnected pathways.
? Liver is the major site of amino acid degradation in
vertebrates.
? PLP facilitates the transamination and other
transformations of amino acids.
? Glutamate collects and delivers free ammonia to
the liver.
? Gln and Glu releases NH4+ in liver mitochondria.
? NH4+ in hepatocytes is converted into urea through
the urea cycle in most terrestrial vertebrates for
excretion.
? The conversion of ammonia to urea takes five (six)
enzymatic steps.
? The rate of urea synthesis is controlled at two
levels.
? The carbon skeletons of the amino acids are first
converted into seven major metabolic
intermediates.
? Some amino acids are converted to intermediates
of citric acid cycle by simple removal of the amino
groups.
? Acetyl-CoA is formed from the degradation of
many amino acids.
? O2 is used to break the aromatic rings of Pro,
Phe and Tyr,as well as to oxidize Cys.
? A few genetic diseases are related to defects of
Phe catabolism enzymes.
? Leu,Ile,and Val are degraded via reactions
similar to fatty acid oxidation.
