Bài giảng Biochemistry 2/e - Chapter 26: Nitrogen Acquisition and Amino Acid Metabolism

Chapter 26Nitrogen Acquisition and Amino Acid Metabolismto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline26.1 The Two Major Pathways of N Acquisition 26.2 The Fate of Ammonium 26.3 Glutamine Synthetase 26.4 Amino Acid Biosynthesis 26.5 Metabolic Degradation of Amino Acids Major Pathways for N AcquisitionAll biological compounds contain N in a reduced form The principal inorganic forms of N are in an oxidized state Thus, N acquisition must involve reduction of the oxidized forms (N2 and NO3-) to NH4+ Nearly all of this is in microorganisms and green plants. Animals gain N through diet. Overview of N AcquisitionNitrogen assimilation and nitrogen fixation Nitrate assimilation occurs in two steps: 2e- reduction of nitrate to nitrite and 6e- reduction of nitrite to ammonium (page 854) Nitrate assimilation accounts for 99% of N acquisition by the biosphere Nitrogen fixation involves reduction of N2 in prokaryotes by nitrogenase Nitrate AssimilationElectrons are transferred from NADH to nitrate Pathway involves -SH of enzyme, FAD, cytochrome b and MoCo - all protein-bound Nitrate reductases are big - 210-270 kD See Figure 26.2 for MoCo structure MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer Nitrite ReductaseLight drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite See Figure 26.2b for siroheme structure Nitrite is reduced to ammonium while still bound to siroheme In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolicEnzymology of N fixationOnly occurs in certain prokaryotes Rhizobia fix nitrogen in symbiotic association with leguminous plants Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates All nitrogen fixing systems appear to be identical They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits and NH4+ inhibits expression of nif genes Nitrogenase ComplexTwo protein components: nitrogenase reductase and nitrogenase Nitrogenase reductase is a 60 kD homodimer with a single 4Fe-4S cluster Very oxygen-sensitive Binds MgATP 4ATP required per pair of electrons transferred Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2Why should nitrogenase need ATP???N2 reduction to ammonia is thermodynamically favorable However, the activation barrier for breaking the N-N triple bond is enormous 16 ATP provide the needed activation energy NitrogenaseA 220 kD heterotetramer Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc) Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor Nitrogenase is slow - 12 e- pairs per second, i.e., only three molecules of N2 per second The Fate of AmmoniumThree major reactions in all cells Carbamoyl-phosphate synthetase Itwo ATP required - one to activate bicarb, one to phosphorylate carbamate Glutamate dehydrogenase reductive amination of alpha-ketoglutarate to form glutamate Glutamine synthetase ATP-dependent amidation of gamma-carboxyl of glutamate to glutamine Ammonium AssimilationTwo principal pathways Principal route: GDH/GS in organisms rich in N See Figure 26.11 - both steps assimilate N Secondary route: GS/GOGAT in organisms confronting N limitation GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase See Figures 26.12 and 26.13Glutamine SynthetaseA Case Study in RegulationGS in E. coli is regulated in three ways:Feedback inhibitionCovalent modification (interconverts between inactive and active forms)Regulation of gene expression and protein synthesis control the amount of GS in cellsBut no such regulation occurs in eukaryotic versions of GSAllosteric Regulationof Glutamine SynthetaseNine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-PGly, Ala, Ser are indicator of amino acid metabolism in cellsOther six are end products of a biochemical pathwayThis effectively controls glutamine’s contributions to metabolismCovalent Modificationof Glutamine SynthetaseEach subunit is adenylylated at Tyr-397Adenylylation inactivates GSAdenylyl transferase catalyzes both the adenylylation and deadenylylationPII (regulatory protein) controls theseAT:PIIA catalyzes adenylylationAT:PIID catalyzes deadenylylation-ketoglutarate and Gln also affectGene Expressionregulates GSGene GlnA is actively transcribed only if transcriptional enhancer NRI is in its phosphorylated form, NRI-PNRI is phosphorylated by NRII, a protein kinaseIf NRII is complexed with PIIA it acts as a phosphatase, not a kinaseAmino Acid BiosynthesisPlants and microorganisms can make all 20 amino acids and all other needed N metabolites In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions Mammals can make only 10 of the 20 aas The others are classed as "essential" amino acids and must be obtained in the diet All amino acids are grouped into families according to the intermediates that they are made from - see Table 26.1The -Ketoglutarate FamilyGlu, Gln, Pro, Arg, and sometimes Lys Proline pathway is chemistry you have seen before in various ways Look at ornithine pathway to see the similarity to the proline pathway Note that CPS-I converts ornithine to citrulline in the urea cycle (Figure 26.23) Know the CPS-I mechanism - Figure 26.22The Urea CycleN and C in the guanidino group of Arg come from NH4+, HCO3- (carbamoyl-P), and the -NH2 of Glu and AspBreakdown of Arg in the urea cycle releases two N and one C as ureaImportant N excretion mechanism in livers of terrestrial vertebratesUrea cycle is linked to TCA by fumarateLysine Biosynthesisin some fungi and in EuglenaLys derived from -ketoglutarateMust add one C - it’s done as in TCA!Transamination gives -aminoadipateAdenylylation activates the -COOH for reductionReductive amination give saccharopineOxidative cleavage yields lysineThe Aspartate FamilyAsp, Asn, Lys, Met, Thr, IleTransamination of OAA gives AspAmidation of Asp gives AsnThr, Met, and Lys are made from Asp (See Figure 26.27)-Aspartyl semialdehyde and homoserine are branch pointsNote role of methionine in methylations via S-adenosylmethionine (Fig. 26.28)The Pyruvate FamilyAla, Val, LeuTransamination of pyruvate gives AlaVal is derived from pyruvateNote that Ile synthesis from Thr mimics Val synthesis from pyruvate (Fig. 26.29)Leu synthesis, like that of Ile and Val, begins with an -keto acidTransaminations from Glu complete each of these pathways (Figs. 26.29-30)3-Phosphoglycerate FamilySer, Gly, Cys3-Phosphoglycerate dehydrogenase diverts 3-PG from glycolysis to aa pathsTransamination by Glu gives 3-P-serinePhosphatase yields serineSerine hydroxymethylase (PLP) transfers the -carbon of Ser to THF to make glycineA PLP-dependent enzyme makes CysAromatic Amino AcidsPhe, Tyr, Trp, HisShikimate pathway yields Phe, Tyr, TrpNote the role of chorismate as a branch point in this pathway (Figs. 26.36-7)Note the ‘channeling’ in tryptophan synthase (Figure 26.39)His synthesis, like that of Trp, shares metabolic intermediates with purine biosynthetic pathwayDegradation of Amino AcidsThe 20 amino acids are degraded to produce (mostly) TCA intermediates Know the classifications of amino acids in Figure 26.41 Know which are glucogenic and ketogenic Know which are purely ketogenic