第第 1212 章 氨基酸代谢章 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 acids

Degradation & absorption of dietary proteins

Pepsin: the first enzymediscovered (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

R C H C O O— —-|

N H 3+

R C C O O— —-

N H 3

+

|R C C O O + H— —

- +|O|

FA DF M N（ ）

FA D HF M N H

2（ ）2

H O2 N H 3

H O2 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

N H 3+

|H C C O O— —

-

|C H 2|

C H 2|

C O O-

+ N A D P（ ）+

+ H O2 N H + 4+

+ N A D P H （ ） + H+

O| |C C O O—

-

|C H 2|

C H 2|

C O 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

R 1|

H C N H— — 2|C O O H

R 2|

C = O|C O O H

+

R 1|

H C N H— — 2|C O O H

R 2|

C = O|C O O H

+

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

C H 3|

H C N H— — 2|C O O H

H O —

H C3 —

H C = O|

O| |

|O

-

— — — —C H O P O2-

C H 3|

C = O|C O O H N

H O —

H C3 —

C H N H2— 2|

O| |

|O

-

— — — —C H O P O2

-

（ ）C H 2 2

C O O H|

|C H N H— 2|

C O O H

（ ）C H 2 2

C O O H|

|C = O

|C O 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

二二 .. 脱羧基作脱羧基作用用

N H 2|

R C H C O O H — — R C H— 2

N H 2|氨 基 酸 脱 羧 酶

P L P+ C O 2

PLP acts as a temporarycarrier of amino groups

at the active sites ofall aminotransferases.

PLP facilitates several different types of

transformation aroundthe -carbon of

amino acids.

PLP is derived fromvitamin 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 谷氨酸＋丙酮酸 α-α- 酮戊二酸＋丙氨酸酮戊二酸＋丙氨酸

谷氨酸＋草酰乙酸 谷氨酸＋草酰乙酸 α-α- 酮戊二酸＋天冬氨酸酮戊二酸＋天冬氨酸

三 . 氨基酸碳架的分解

N H 3+

|H C C O O— —

-

|C H 2|

C H 2|

C O O-

+ N A D P（ ）+

+ H O2N H + 4+

+ N A D P H （ ） + H+

O| |C C O O—

-

|C H 2|

C H 2|

C O O-

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

N RN O 3

- ＋ 2 H ＋ 2 e- N O 2

- ＋ H O 2

N iRN O 2

-＋ 1 2 H

+ ＋ 6 e- N H 4

+ ＋ 2 H O2

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

-+2ATPH2N-CO-O-PO3-+2ADP+Pi+2H+

N-acetylglutamate is an essential allosteric activator for this enzyme

第四节 Ammonium assimilation

The synthesis ofCarbamoyl （氨甲酰） phosphate requires

two activationsteps, consuming twoATP molecules: onefor activating HCO3

-,the other to

phosphorylatecarbamate.

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 -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 synthetasehas 12 subunits arranged as two

rings of hexamers.

Activesites

Tyr397

(adenylylation site)

The glutamine synthetase is accumulativelyinhibited by at least 8 allostericeffectors, mostly end productsof glutaminemetabolism.

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 (22)or FeMo protein

Reductase: a dimer of two Identical subunits bridged by a 4Fe-4S. ATP hydrolysis is coupled to protein conformatinal changes.

Dinitrogenasereductase (dimer)or Fe protein

ADP

ADP

4Fe-4S

8Fe-7S(P-cluster)

Fe-Mo cofactor e-

Fe-Mo cofactor

8Fe-7S(P-cluster)

4Fe-4SADP

ADP

Molybdenum (or vanadium)

N2 is believed to be reduced by theFe-Mo cofactorN2

Fe

FeFe

Fe

FeFe Fe

SS

S

S

S

S

SSS

Mo

高柠檬酸

Electrons are transferred

through a series of carriers

to N2 for its reduction on

the nitrogenase complex.

Electrons are transferredto N2 bound in the active site of dinitrogenasevia ferredoxin/flavodoxin and dinitrogenaseReductase.

N2 + 8H+ +8e- + 16ATP + 16H2O 2NH3 + H2 + 16ADP + 16Pi

(or photophosphorylation)

Conformational changereduces 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.

GluDehydrogenase

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.