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Chapter 26

Chapter 26. Nitrogen Acquisition and Amino Acid Metabolism to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 26

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  1. Chapter 26 Nitrogen Acquisition and Amino Acid Metabolism to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All 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

  2. Outline • 26.1 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

  3. Major Pathways for N Acquisition • All 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.

  4. Overview of N Acquisition Nitrogen 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

  5. Nitrate Assimilation Electrons 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

  6. Nitrite Reductase Light 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 cytosolic

  7. Enzymology of N fixation Only 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

  8. Nitrogenase Complex Two 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 N2

  9. Why 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

  10. Nitrogenase A 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

  11. The Fate of Ammonium Three major reactions in all cells • Carbamoyl-phosphate synthetase I • two 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

  12. Ammonium Assimilation Two 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 26.13

  13. Glutamine SynthetaseA Case Study in Regulation • GS in E. coli is regulated in three ways: • Feedback inhibition • Covalent modification (interconverts between inactive and active forms) • Regulation of gene expression and protein synthesis control the amount of GS in cells • But no such regulation occurs in eukaryotic versions of GS

  14. Allosteric Regulationof Glutamine Synthetase • Nine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-P • Gly, Ala, Ser are indicator of amino acid metabolism in cells • Other six are end products of a biochemical pathway • This effectively controls glutamine’s contributions to metabolism

  15. Covalent Modificationof Glutamine Synthetase • Each subunit is adenylylated at Tyr-397 • Adenylylation inactivates GS • Adenylyl transferase catalyzes both the adenylylation and deadenylylation • PII (regulatory protein) controls these • AT:PIIA catalyzes adenylylation • AT:PIID catalyzes deadenylylation • -ketoglutarate and Gln also affect

  16. Gene Expressionregulates GS • Gene GlnA is actively transcribed only if transcriptional enhancer NRI is in its phosphorylated form, NRI-P • NRI is phosphorylated by NRII, a protein kinase • If NRII is complexed with PIIA it acts as a phosphatase, not a kinase

  17. Amino Acid Biosynthesis • Plants 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 26.1

  18. The -Ketoglutarate Family Glu, 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 26.22

  19. The Urea Cycle • N and C in the guanidino group of Arg come from NH4+, HCO3- (carbamoyl-P), and the -NH2 of Glu and Asp • Breakdown of Arg in the urea cycle releases two N and one C as urea • Important N excretion mechanism in livers of terrestrial vertebrates • Urea cycle is linked to TCA by fumarate

  20. Lysine Biosynthesisin some fungi and in Euglena • Lys derived from -ketoglutarate • Must add one C - it’s done as in TCA! • Transamination gives -aminoadipate • Adenylylation activates the -COOH for reduction • Reductive amination give saccharopine • Oxidative cleavage yields lysine

  21. The Aspartate FamilyAsp, Asn, Lys, Met, Thr, Ile • Transamination of OAA gives Asp • Amidation of Asp gives Asn • Thr, Met, and Lys are made from Asp (See Figure 26.27) • -Aspartyl semialdehyde and homoserine are branch points • Note role of methionine in methylations via S-adenosylmethionine (Fig. 26.28)

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