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Nitrogen Acquisition & Amino Acid Metabolism Overview

Explore the major pathways of nitrogen acquisition and amino acid metabolism, including nitrate assimilation and nitrogen fixation. Learn about key enzymes and regulatory controls in nitrogen metabolism. Understand the roles of glutamine synthetase and nitrogenase in cellular processes. Delve into the fate of ammonium and pathways of ammonium assimilation. Gain insights into the complex mechanisms governing nitrogen utilization in biological systems.

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Nitrogen Acquisition & Amino Acid Metabolism Overview

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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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