Biochemistry 432/832

1. Biochemistry 432/832 February 12, 2002 Chapter 26 Nitrogen Acquisition

2. Announcements: -

3. Outline • 26.1The 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

4. 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.

5. The nitrogen cycle

6. 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 • Nitrate assimilation accounts for 99% of N acquisition by the biosphere • Nitrogen fixation involves reduction of N2 in prokaryotes by nitrogenase

7. 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 kDa • MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer

8. Novel prosthetic groups used in N acquisition Molybdopterin Siroheme

9. Mo-containing enzymes Mo is the heaviest element used by eukaryotes Two classes of molybdoenzymes 1) Molybdopterin-dependent enzymes Nitrate reductase Formate dehydrogenase Aldehyde oxidase Xanthine dehydrogenase Sulfate oxidase 2) Nitrogenase Molybdopterin

10. Nitrite Reductase Light drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite • Nitrite is reduced to ammonium while still bound to siroheme • In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolic

12. Enzymology of N fixation Only occurs in certain prokaryotes • Rhizobia fix nitrogen in symbiotic association with plants • Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates • All nitrogen fixing systems are very similar • They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits reaction and NH4+ inhibit expression of nif genes)

13. Nitrogenase Complex Two protein components: nitrogenase reductase and nitrogenase • Nitrogenase reductase is a 60 kDa 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

14. 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

15. To break the triple bond, energy input in necessary

16. Nitrogenase A 220 kDa 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

17. Structures of two types of metal clusters in nitrogenase: The P-cluster FeMoCo

18. The nitrogenase reaction Accumulation of electrons

19. Complex between nitrogenase reductase and nitrogenase Nitrogenase reductase

20. Regulation of nitrogen fixation ADP inhibits NH4+ represses expression ADP-ribosylation inhibits

21. The Fate of Ammonium Three major reactions in all cells • Carbamoyl-phosphate synthetase • two ATP required - one to activate bicarbonate, 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

22. The glutamate dehydrogenase reaction

23. The glutamine synthetase reaction

24. Ammonium Assimilation Two principal pathways • Principal route: GDH/GS in organisms rich in N • both steps assimilate N • Secondary route: GS/GOGAT in organisms confronting N limitation • GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase

25. The glutamate dehydrogenase/glutamine synthase pathway Two N fixing steps - one inefficient One each

26. The glutamate synthase reaction

27. The glutamine synthase/GOGAT pathway One N fixing step - inefficient but expensive One NADPH Two ATP

28. 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

29. The glutamine synthetase reaction

30. Glutamine synthetase structure stack of two hexagons

31. 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 indicators of amino acid metabolism in cells • Other six are end products of biochemical pathways • This effectively controls glutamine’s contributions to metabolism

32. Allosteric regulation of glutamine synthase activity by feedback inhibition

33. 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 both activities • AT:PIIA catalyzes adenylylation • AT:PIID catalyzes deadenylylation • -ketoglutarate and Gln also affect

34. Covalent modification of glutamine synthase - adenylylation of Tyr397