南京农业大学生物化学课件29.ppt

南京农业大学生物化学课件29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 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 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 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 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程，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。叫氧化脱氨基作用。一一.氨的去氨的去路路 Glu +NAD(P)+H2O a-KG+NH4+NADH(P)+H+2.脱氢酶作用-GDH一一.氨的去氨的去路路 3.转氨基作用一一.氨的去氨的去路路 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）的催化作的催化作用转移到用转移到-酮酸的羰基上，结果生成新的酮酸，而原来的酮酸的羰基上，结果生成新的酮酸，而原来的-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.氨的去氨的去路路 4.联联合合脱脱氨氨作作用用(转转氨氨酶酶-谷谷氨氨酸酸脱脱氢氢酶酶)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.氨的去氨的去路路 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 二二二二.脱羧基作脱羧基作脱羧基作脱羧基作用用用用 PLPPLP acts as a temporary acts as a temporarycarrier of amino groupscarrier of amino groupsat the active sites ofat the active sites ofall all aminotransferases.aminotransferases.PLP facilitates several PLP facilitates 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 aminotransferases have been Serum aminotransferases have been used as clinical markers of tissue used as clinical markers of tissue damagesdamagesDamaged 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 谷氨酸丙谷氨酸丙谷氨酸丙谷氨酸丙酮酮酮酮酸酸酸酸 -酮酮酮酮戊二酸丙氨酸戊二酸丙氨酸戊二酸丙氨酸戊二酸丙氨酸谷氨酸草谷氨酸草谷氨酸草谷氨酸草酰酰酰酰乙酸乙酸乙酸乙酸 -酮酮酮酮戊二酸天冬氨酸戊二酸天冬氨酸戊二酸天冬氨酸戊二酸天冬氨酸三三.氨基酸碳架的分解氨基酸碳架的分解 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，大多数氨基例如，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。糖及酮体的含量同时增加。在体内可以转变为糖的氨在体内可以转变为糖的氨基酸称为基酸称为生糖氨基酸生糖氨基酸，按糖代谢途径进行代谢；能转，按糖代谢途径进行代谢；能转变为酮体的氨基酸称为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架的分解 硝硝酸酸盐盐还还原原分分两两步步进进行行：第第一一步步在在硝硝酸酸还还原原酶酶（nitrate reductase,NR）催催化化下下，由由NAD（P）H提提供供1对对电电子子，硝硝酸酸盐盐被被还还原原为为亚亚硝硝酸酸盐盐，第第二二步步是是在在亚亚硝硝酸酸还还原原酶酶（nitrite reductase,NiR）下下，由由还还原原型型铁铁氧氧还还蛋蛋白白（Fdred）提提供供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 硝硝酸酸盐盐还还原原分分两两步步进进行行：第第一一步步在在硝硝酸酸还还原原酶酶（nitrate reductase,NR）催催化化下下，由由NAD（P）H提提供供1对对电电子子，硝硝酸酸盐盐被被还还原原为为亚亚硝硝酸酸盐盐，第第二二步步是是在在亚亚硝硝酸酸还还原原酶酶（nitrite reductase,NiR）下下，由由还还原原型型铁铁氧氧还还蛋蛋白白（Fdred）提提供供3对对电电子子，使使亚亚硝硝酸酸盐盐（NO2-）还原成氨。还原成氨。第三节第三节 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(氨氨甲甲酰酰磷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 hepatocytes(in 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 vertebratesUreaUrea is formed from ammoniaammonia,COCO2 2(as bicarbonate)and AspAsp.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第四节第四节 Ammonium assimilationThe synthesis ofThe synthesis ofCarbamoyl Carbamoyl（氨甲酰）氨甲酰）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 phosphorylatephosphorylatecarbamate.carbamate.an anhydride1.Carbamoyl-phosphate synthetase I 该该反反应应消消耗耗2 2个个ATPATP分分子子中中的的两两个个高高能能磷磷酸酸键键，其其中中1 1个个是是用用于于活活化化HCOHCO3 3-，另另1 1分分子子ATPATP则则用用于于磷磷酸化氨甲酰基。酸化氨甲酰基。第四节第四节 Ammonium assimilationFumarate is converted back to Fumarate is converted back to Asp via a partial usage of the Asp via a partial usage of the citric acid cycle.citric acid cycle.The rate of urea synthesis is The rate of urea synthesis is controlled at two levelscontrolled at two levelsAllosteric Allosteric（别构）别构）regulationregulation:N-acetylglutamate,by binding to a site which hydrolyzes（水解）Gln in another isozyme,positively regulates carbamoyl phosphate synthetase 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 defects of the urea cycle Genetic defects of the urea cycle enzymes lead to hyperammonemia enzymes lead to hyperammonemia and brain damageand brain damageHigh 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 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 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 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第四节第四节 Ammonium 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+.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 synthetase is a The Glutamine synthetase 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 allosteric effectors and reversible allosteric effectors and reversible adenylylation.adenylylation.The bacterialThe bacterial glutamine synthetaseglutamine synthetasehas 12 subunits arranged as twohas 12 subunits arranged as tworings of hexamers.rings of hexamers.Activesites Tyr397(adenylylation site)The glutamine The glutamine synthetase is synthetase is accumulativelyaccumulativelyinhibited by at inhibited by at least 8 allostericleast 8 allostericeffectors,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)Reductant+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 dinitrogenase(The dinitrogenase(固氮酶固氮酶)complex complex in certain bacteria(diazotrophs)in certain bacteria(diazotrophs)catalyzes the conversion of Ncatalyzes the conversion of N2 2(azoteazote,“without life”)to NH“without life”)to NH3 3,which is,which is the ultimate source of nitrogen for all the ultimate source of nitrogen for all nitrogen-containing biomolecules.nitrogen-containing biomolecules.N2+8H+8e-2NH3+H2 The Haber method:N2+3H2 2NH3 Go=-33.5kJ/mol with iron catalyst,500oC,300 atmospheres.The nitrogenase complex consists of The nitrogenase complex consists of dinitrogenasedinitrogenase and and dinitrogenase redutasedinitrogenase redutase both being iron-sulfur proteins.both being iron-sulfur proteins.Dinitrogenase(Dinitrogenase(a a2 2b b2 2)or FeMo proteinor FeMo proteinReductase:a dimer of two Reductase:a dimer of two Identical subunits bridged Identical subunits bridged by a 4Fe-4S.by a 4Fe-4S.ATP hydrolysis is coupled to ATP hydrolysis is coupled to protein conformatinal changes.protein conformatinal changes.DinitrogenaseDinitrogenasereductase(dimer)reductase(dimer)or Fe proteinor Fe proteinADPADP4Fe-4S8Fe-7S(P-cluster)Fe-Mo cofactor e-Fe-Mo cofactor8Fe-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 nitrogenase reduction on the nitrogenase plex.Electrons are Electrons are transferredtransferredto Nto N2 2 bound in bound in the active site the active site of dinitrogenaseof dinitrogenasevia ferredoxin/via ferredoxin/flavodoxin and flavodoxin and dinitrogenasedinitrogenaseReductase.Reductase.N2+8H+8e-+16ATP+16H2O 2NH3+H2+16ADP+16Pi(or photophosphorylation)or photophosphorylation)Conformational changeConformational changereduces e-affinityreduces e-affinityThe oxidized dinitrogenase reductase dissociates from the dinitrogenaseReduced dinitrogenase reductase associates with the dinitrogenaseThe nitrogenase complex is The nitrogenase complex is extremely labile to Oextremely labile to O2 2 and various and various protective mechanisms have protective mechanisms have evolved:living anaerobically,evolved:living anaerobically,forming thick walls,uncoupling eforming thick walls,uncoupling e-transport from ATP synthesis transport from ATP synthesis(entering O(entering O2 2 is used inmediately)or is used inmediately)or being protected by Obeing protected by O2 2-binding-binding proteins.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 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 synthetase pathway:glutamine synthetase and glutamate synthaseand glutamate synthase (an enzyme only present in bacteria and plants).Gln Gln synthetasesynthetaseGluGluSynthaseSynthase(+NADPH(+NADPH+ATP)+ATP)Gln Gln synthetasesynthetaseThe pathways for ammonia The pathways for ammonia to enter organic compounds.to enter organic compounds.GluGluDehydrogenaseDehydrogenaseVery minor)Very minor)AsnAsnsynthetasesynthetase Carbamoyl Carbamoyl Phosphate Phosphate SynthetaseSynthetase TransaminationTransamination(or NHor NH4 4+)SummarySummaryAmino 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.谢谢观赏谢谢观赏