南京农业大学生物化学课件29

1、第一节第一节 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 incorp

2、oration 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.第一节第一节 The nitrogen cycle The formation of NH4+ from N2 gas is termed nitrogen fixation. N2 fix

3、ation 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 o

4、f microbial decomposition following death.第一节第一节 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 t

5、he metabolic activity of denitrifying bacteria. 第一节第一节 The nitrogen cycle Dietary proteins are digested into amino acids in the gastrointestinal(胃胃肠肠) tract via the action of pepsin, trypsin, chymotrypsin, carboxypeptidases and aminopeptidases.Sources of amino acids for animalsSources of amino acids

6、 for animalsProteins (but not pepsin) unfoldedAbsorbed as tri- & dipeptides,and amino acidsDegradation & absorption Degradation & absorption of dietary proteinsof dietary proteinsPepsin: the first enzymediscovered (18th century).proteasesEssential amino acidsEssential amino acidsAmino ac

7、ids 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 acidsOverall fate of excess amino acids第二节第二节 Amino acid degradation 1. 氧化脱氨基氧化脱氨基 氨基酸在酶的作用下脱去氨基生成相应酮酸的过氨基酸在酶的作用下脱去氨基生成相应酮酸

8、的过程，叫氧化脱氨基作用。程，叫氧化脱氨基作用。一一. 氨的去路氨的去路 RCHCOO-|NH3+RCCOO -NH3+|RCCOO + H - +|O|FADFMN（）FADHFMNH2（）2H O2NH3H O22O2氨基酸氧化酶Glu + NAD(P) + H2O a-KG + NH4+ + NADH(P) + H+ 2. 脱氢酶作用-GDH一一. 氨的去路氨的去路 NH3+|HCCOO -|CH2|CH2|COO-+ NADP（ ）+ + H O2NH + 4+ NADPH （ ）+ H+O|CCOO-|CH2|CH2|COO-L- 谷氨酸 -酮戊二酸谷氨酸脱氢酶 3. 转氨基作用一一

9、. 氨的去路氨的去路 R1|HCNH 2|COOHR2|C=O|COOH+R1|HCNH 2|COOHR2|C=O|COOH+转氨酶 转氨基作用是转氨基作用是-氨基酸和氨基酸和-酮酸之间氨基的转移作用酮酸之间氨基的转移作用。一种。一种-氨基酸的氨基酸的-氨基借助转氨酶（氨基借助转氨酶（transaminase）的催的催化作用转移到化作用转移到-酮酸的羰基上，结果生成新的酮酸，而原酮酸的羰基上，结果生成新的酮酸，而原来的来的-酮酸则形成相应的氨基酸。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谷丙转氨酶催化的转氨基作用机理 一一. 氨的去路氨的去路 CH3|HCNH

10、2|COOHHOH C3HC=O|O|O- CHOPO2-CH3|C=O|COOHNHOH C3CHNH22|O|O- CHOPO2-（）CH22COOH|CHNH2|COOH（）CH22COOH|C=O|COOH丙氨酸 磷酸吡哆醛 谷氨酸丙酮酸磷酸吡哆胺 -酮戊二酸 4. 联合脱氨作用联合脱氨作用(转氨酶转氨酶-谷氨酸脱氢酶谷氨酸脱氢酶)谷氨酸谷氨酸 -酮戊二酸 丙丙氨酸氨酸 丙酮酸丙酮酸 转氨酶转氨酶 谷氨酸脱氢酶谷氨酸脱氢酶 NAD(P)+H+ NAD(P)+ 联合脱氨基作用联合脱氨基作用 PLP 一一. 氨的去路氨的去路 在氨基酸脱羧酶催化下进行脱羧作用，生成一个在氨基酸脱羧酶催化下进

11、行脱羧作用，生成一个伯胺类化合物和伯胺类化合物和CO2，其反应可以用下式表示其反应可以用下式表示 NH2|RCHCOOH RCH2NH2|氨基酸脱羧酶PLP+ CO2PLPPLP acts as a temporary acts as a temporarycarrier of amino groupscarrier of amino groupsat the active sites ofat the active sites ofall all aminotransferasesaminotransferases. .PLP facilitates several PLP facilita

12、tes several different types of different types of transformation aroundtransformation aroundthe the a a-carbon of-carbon ofamino acids.amino acids.PLPPLP is derived from is derived fromvitamin Bvitamin B6 6 (pyridoxine (pyridoxine，吡哆醇吡哆醇) )吡哆醛磷酸吡哆醛磷酸磷酸吡哆胺磷酸吡哆胺Serum Serum aminotransferasesaminotransf

13、erases have been have been used as clinical markers of tissue used as clinical markers of tissue damagesdamages Damaged heart or liver cells leak aminotransferases. Blood aspartate aminotransferase and alanine aminotransferase are usually examined for indications of illness.三三. 氨基酸碳架的分解氨基酸碳架的分解 氨基酸脱

14、羧酶 1. 1. 进入进入TCATCA循环循环 Oxidation of the Oxidation of the carbon skeletons of carbon skeletons of amino acids in amino acids in mammalsmammals2.2.再合成为氨基酸再合成为氨基酸 三三. 氨基酸碳架的分解氨基酸碳架的分解 NH3+|HCCOO -|CH2|CH2|COO-+ NADP（ ）+ + H O2NH+ 4+ NADPH （ ）+ H+O|CCOO-|CH2|CH2|COO-3.3.转变为糖和脂肪转变为糖和脂肪 当体内不需要将当体内不需要将-酮酸

15、再合成氨基酸，并且体酮酸再合成氨基酸，并且体内的能量供给充足时，内的能量供给充足时，-酮酸可以转变为糖或脂肪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。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。萄糖及酮体的含量同时增加。在体内可以转变为糖的在体内可以转变为糖的氨基酸称为氨基酸称为生糖氨基酸生糖氨基酸，按糖代谢途径进行代谢；能，按糖代谢途径进行代谢；能转变为酮体的氨基酸称为转变为酮体的氨基酸称为生酮氨基酸。生酮氨基酸。 三三. 氨基酸碳架的分解氨基酸碳

16、架的分解 硝酸盐还原分两步进行：第一步在硝酸盐还原分两步进行：第一步在硝酸还原酶硝酸还原酶（nitrate reductase, NR）催化下，由催化下，由NAD（P）H提供提供1对对电子，硝酸盐被还原为亚硝酸盐，第二步是在亚硝酸还原电子，硝酸盐被还原为亚硝酸盐，第二步是在亚硝酸还原酶（酶（nitrite reductase, NiR）下，由还原型铁氧还蛋白下，由还原型铁氧还蛋白（Fdred）提供提供3对电子，使亚硝酸盐（对电子，使亚硝酸盐（NO2-）还原成氨。还原成氨。 第三节第三节 Nitrate reduction NRNO3- 2H 2e- NO2- H O 2NiRNO2 - 12H

17、+ 6e- NH4+ 2H O2 硝酸盐还原分两步进行：第一步在硝酸盐还原分两步进行：第一步在硝酸还原酶硝酸还原酶（nitrate reductase, NR）催化下，由催化下，由NAD（P）H提供提供1对电子，硝酸盐被还原为亚硝酸盐，第二步是在亚硝对电子，硝酸盐被还原为亚硝酸盐，第二步是在亚硝酸还原酶（酸还原酶（nitrite reductase, NiR）下，由还原型铁氧下，由还原型铁氧还蛋白（还蛋白（Fdred）提供提供3对电子，使亚硝酸盐（对电子，使亚硝酸盐（NO2-）还原成氨。还原成氨。 第三节第三节 Nitrate reduction Ammonium enters organic

18、 linkage via three major reactions that are found in all cells. The enzymes mediating these reactions are：(1) Cabamoyl-phosphate synthetase I (氨甲酰磷氨甲酰磷酸合成酶酸合成酶)(2) Glutamate dehydrogenase（谷氨酸脱氢酶）谷氨酸脱氢酶）, (3) Glutamine synthetase（谷氨酰氨合成酶）谷氨酰氨合成酶）. 第四节第四节 Ammonium assimilationNHNH4 4+ + in in hepatocy

19、tes hepatocytes ( (肝细胞肝细胞) ) is convert ed is convert ed into urea for excretion via the urea cycle in into urea for excretion via the urea cycle in most terrestrial vertebratesmost terrestrial vertebrates UreaUrea is formed from ammoniaammonia, COCO2 2 (as bicarbonate) and AspAsp. The pathway was a

20、lso 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 am

21、monium, 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第四节第四节 Ammonium assimilationThe synthesis ofThe synthesis ofCarbamoylCarbamoyl （氨甲酰）氨

22、甲酰） phosphate phosphate requires requires two activationtwo activationsteps, consuming steps, consuming twotwoATPATP molecules: one molecules: onefor activating HCOfor activating HCO3 3- -, ,the other to the other to phosphorylatephosphorylatecarbamatecarbamate. . an anhydride1. Carbamoyl-phosphate

23、synthetase I 该反应消耗该反应消耗2 2个个ATPATP分子中的两个高能磷酸键分子中的两个高能磷酸键，其中，其中1 1个是用于活化个是用于活化HCOHCO3 3- -，另另1 1分子分子ATPATP则用于则用于磷酸化氨甲酰基。磷酸化氨甲酰基。 第四节第四节 Ammonium assimilationFumarateFumarate is converted back to is converted back to Asp via a partial usage of the Asp via a partial usage of the citric acid cycle.citri

24、c acid cycle.The rate of urea synthesis is The rate of urea synthesis is controlled at two levelscontrolled at two levels AllostericAllosteric （别构）别构）regulationregulation: N-acetylglutamate, by binding to a site which hydrolyzes （水解） Gln in another isozyme, positively regulates carbamoyl phosphate s

25、ynthetase I activity. Gene regulationGene 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 d

26、efects of the urea cycle Genetic defects of the urea cycle enzymes lead to enzymes lead to hyperammonemiahyperammonemia and brain damageand 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 base

27、d on a thorough understanding of the underlying biochemistry. Strategy IStrategy I: diet control, provide the essential amino acids in their a-keto acid forms. Strategy IIStrategy II: when argininosuccinate lyase is deficient, ingesting a surplus of Arg will help (ammonia will be carried out of the

28、body in the form of argininosuccinate, instead of urea). Strategy IIIStrategy 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 e

29、xcreted 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第四节第四节 Ammon

30、ium assimilationThe glutamate dehydrogenase reaction第四节第四节 Ammonium assimilation3. 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+. Glutam

31、ine 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.第四节第四节 Ammonium assimilation谷氨酰胺合成酶谷氨酰胺合成酶第四节第四节 Ammonium assimilationThe Glutamine The Glutamine syntheta

32、sesynthetase is a is a primary regulatory point in primary regulatory point in nitrogen metabolism: being nitrogen metabolism: being regulated by at least eight regulated by at least eight allostericallosteric effectors and reversible effectors and reversible adenylylationadenylylation. . The bacter

33、ialThe bacterial glutamine glutamine synthetasesynthetasehas 12 subunits arranged as twohas 12 subunits arranged as tworings of rings of hexamershexamers. .Activesites Tyr397(adenylylation site)The glutamine The glutamine synthetasesynthetase is is accumulativelyaccumulativelyinhibited by at inhibit

34、ed by at least 8 least 8 allostericallostericeffectors, mostly effectors, mostly end productsend productsof glutamineof glutaminemetabolism.metabolism.Glutamate synthase catalyes the reductive amination of a-ketoglutarate suing the amide-N of glutamine as the N donor:Glutamate synthase (GOGAT) Reduc

35、tant +a-KG+Gln 2 Glu+oxidized redctant 第四节第四节 Ammonium assimilationThe glutamate synthase reaction谷氨酸合酶第四节第四节 Ammonium assimilationOnly certain bacteria can fix NOnly certain bacteria can fix N2 2 into ammonia into ammoniaRhizobiaCyanobacteria蓝细菌根瘤菌第第5节节 Nitrogen fixationThe The dinitrogenase dinitr

36、ogenase ( (固氮酶固氮酶) ) complex complex in certain bacteria (in certain bacteria (diazotrophsdiazotrophs) ) catalyzes the conversion of Ncatalyzes the conversion of N2 2 ( (azoteazote, “without life”) to NH, “without life”) to NH3 3, , which is the ultimate source of which is the ultimate source of nit

37、rogen for all nitrogen-containing nitrogen for all nitrogen-containing biomoleculesbiomolecules. . N2 + 8H+ + 8e- 2NH3 + H2 The Haber method: N2 +3H2 2NH3 Go = - 33.5kJ/mol with iron catalyst, 500oC, 300 atmospheres.The The nitrogenasenitrogenase complex consists of complex consists of dinitrogenase

38、dinitrogenase and and dinitrogenasedinitrogenase redutaseredutase both being iron-sulfur proteins.both being iron-sulfur proteins.DinitrogenaseDinitrogenase ( (a a2 2b b2 2) )or or FeMoFeMo protein proteinReductase: a Reductase: a dimerdimer of two of two Identical subunits bridged Identical subunit

39、s bridged by a 4Fe-4S. by a 4Fe-4S. ATP hydrolysis is coupled to ATP hydrolysis is coupled to protein protein conformatinalconformatinal changes. changes.DinitrogenaseDinitrogenasereductase (reductase (dimerdimer) )or Fe proteinor Fe proteinADPADP4Fe-4S8Fe-7S(P-cluster)Fe-Mo cofactor e-Fe-Mo cofacto

40、r8Fe-7S(P-cluster)4Fe-4SADPADPMolybdenum (or vanadium)N2 is believed to be reduced by theFe-Mo cofactorN2FeFeFeFeFeFeFeSSSSSSSSSMo高柠檬酸高柠檬酸Electrons are transferred through a Electrons are transferred through a series of carriers to Nseries of carriers to N2 2 for its for its reduction on the reducti

41、on on the nitrogenasenitrogenase plex.Electrons are Electrons are transferredtransferredto Nto N2 2 bound in bound in the active site the active site of of dinitrogenasedinitrogenasevia via ferredoxinferredoxin/ /flavodoxinflavodoxin and and dinitrogenasedinitrogenaseReductase.Reductase.N2 + 8H+ +8e

42、- + 16ATP + 16H2O 2NH3 + H2 + 16ADP + 16Pi( (or or photophosphorylationphotophosphorylation) )Conformational changeConformational changereduces e- affinityreduces e- affinityThe oxidized dinitrogenase reductase dissociates from the dinitrogenaseReduced dinitrogenase reductase associates with the din

43、itrogenaseThe The nitrogenasenitrogenase complex is complex is extremely labile to Oextremely labile to O2 2 and various and various protective mechanisms have protective mechanisms have evolved: living evolved: living anaerobicallyanaerobically, , forming thick walls, uncoupling eforming thick wall

44、s, uncoupling e- - transport from ATP synthesis transport from ATP synthesis (entering O(entering O2 2 is used is used inmediatelyinmediately)or )or being protected by Obeing protected by O2 2-binding -binding teins.Genes encoding the protein components of the nitrogenase complex are bei

45、ng transferred into non-nitrogen-fixing bacteria and plants.Reduced nitrogen in the form of Reduced nitrogen in the form of NHNH4 4+ + is assimilated into amino is assimilated into amino acids mainly via a two-enzyme acids mainly via a two-enzyme pathway : glutamine pathway : glutamine synthetasesynthetase and glutamate synthaseand glutamate synthase (an enzyme only present in bacteria and plants).Gln Gln synthetasesynthetaseGluGluSynthaseSynthase(+NADPH(+NADPH+ATP)+ATP)Gl