第12章氨基酸代谢

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

Sources of amino acids for animals Dietary proteins are digested into amino acids in the gastrointestinal(胃肠) tract via the action of pepsin, trypsin, chymotrypsin, carboxypeptidases and aminopeptidases.

Pepsin: the first enzyme discovered (18th century). Proteins (but not pepsin) unfolded proteases Absorbed as tri- & dipeptides, and amino acids Degradation & absorption of dietary proteins 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

一. 氨的去路 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 2. 脱氢酶作用-GDH Glu + NAD(P) + H2O a-KG + NH4++ NADH(P)+ H+

3. 转氨基作用 一. 氨的去路转氨基作用是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 一. 氨的去路谷丙转氨酶催化的转氨基作用机理

4. 联合脱氨作用(转氨酶-谷氨酸脱氢酶) 一. 氨的去路 α-酮戊二酸 NAD(P)+H+ 丙氨酸谷氨酸脱氢酶转氨酶 PLP 丙酮酸 NAD(P)+ 谷氨酸联合脱氨基作用

二.脱羧基作用 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

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. 吡哆醛磷酸 PLP is derived from vitamin B6 (pyridoxine，吡哆醇) 磷酸吡哆胺

Damaged heart or liver cells leak aminotransferases. Blood aspartate aminotransferase and alanine aminotransferase are usually examined for indications of illness. Serum aminotransferases have been used as clinical markers of tissue damages

三. 氨基酸碳架的分解 1. 进入TCA循环氨基酸脱羧酶

Oxidation of the carbon skeletons of amino acids in mammals

三. 氨基酸碳架的分解 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 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。

第三节 Nitrate reduction 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。

第四节 Ammonium assimilation 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）.

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. NH4+ in hepatocytes (肝细胞) is convert ed into urea for excretion via the urea cycle in most terrestrial vertebrates

第四节 Ammonium assimilation 1. Carbamoyl-phosphate synthetase I 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: NH4++HCO3-+2ATPH2N-CO-O-PO3-+2ADP+Pi+2H+ N-acetylglutamate is an essential allosteric activator for this enzyme

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

第四节 Ammonium assimilation 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。

Fumarate is converted back to Asp via a partial usage of the citric acid cycle.

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. The rate of urea synthesis is controlled at two levels

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. Genetic defects of the urea cycle enzymes lead to hyperammonemia and brain damage

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.

第四节 Ammonium assimilation 2. Glutamate dehydrogenase (GDH) Glutamate dehydrogenase catalyzes the reductive amination of a-ketoglutarate to yield glutamate. Reduced pyridine mucleotides (NADH or NADPH) provide the reducing power: 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

The Glutamine synthetase is a primary regulatory point in nitrogen metabolism: being regulated by at least eight allosteric effectors and reversible adenylylation.

The bacterialglutamine 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.

第四节 Ammonium assimilation Glutamate synthase (GOGAT) Glutamate synthase catalyes the reductive amination of a-ketoglutarate suing the amide-N of glutamine as the N donor: Reductant +a-KG+Gln  2 Glu+oxidized redctant

第四节 Ammonium assimilation 谷氨酸合酶 The glutamate synthase reaction

第5节 Nitrogen fixation Only certain bacteria can fix N2 into ammonia Rhizobia Cyanobacteria 蓝细菌根瘤菌

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 Dinitrogenase reductase (dimer) or Fe protein Reductase: a dimer of two Identical subunits bridged by a 4Fe-4S. ATP hydrolysis is coupled to protein conformatinal changes. e- Fe-Mo cofactor ADP 8Fe-7S (P-cluster) ADP 4Fe-4S 4Fe-4S 8Fe-7S (P-cluster) ADP ADP Fe-Mo cofactor

Fe N2 is believed to be reduced by the Fe-Mo cofactor S S S Fe Fe Fe N2 S S S Fe Fe Fe S S S Mo Molybdenum (or vanadium) 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. (or photophosphorylation) Conformational change reduces e- affinity The oxidized dinitrogenase reductase dissociates from the dinitrogenase Reduced dinitrogenase reductase associates with the dinitrogenase N2 + 8H+ +8e- + 16ATP + 16H2O  2NH3 + H2 + 16ADP + 16Pi

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).

The pathways for ammonia to enter organic compounds. ( or NH4+) Gln synthetase Carbamoyl Phosphate Synthetase Glu Dehydrogenase Very minor) Gln synthetase Asn synthetase Glu Synthase (+NADPH +ATP) Transamination

第12章氨基酸代谢