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Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids

Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids. CH353 January 22, 2008. The Nitrogen Cycle. Nitrogen Fixation N 2 → NH 4 + Nitrification NH 3 → NO 2 - → NO 3 - Denitrification NO 2 - & NO 3 - → N 2 Nitrogen Assimilation NH 4 + → Organic nitrogen Deamination

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Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids

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  1. Overview of Nitrogen Metabolismand Biosynthesis of Amino Acids CH353 January 22, 2008

  2. The Nitrogen Cycle • Nitrogen Fixation N2→ NH4+ • Nitrification NH3 → NO2- → NO3- • Denitrification NO2- & NO3- → N2 • Nitrogen Assimilation NH4+ → Organic nitrogen • Deamination Organic nitrogen → NH4+

  3. Nitrogen Fixation • Nitrogen Reduction Reaction N2 + 3 H2→ 2NH3ΔG′º = -33.5 kJ/mol • Exergonic reaction • High activation energy • Stability of N2 triple bond D = 930 kJ/mol • Biological Nitrogen Reduction N2 + 10H+ + 8e- + 16ATP → 2NH4+ + 16ADP + 16Pi + H2

  4. Nitrogenase Complex Dinitrogenase reductase • Dimer (Mr 60,000) of same subunits • One 4Fe-4S between subunits • One ATP/ADP site per subunit • ATP binding enhances reduction potential: E′º -300 → -420 mV Dinitrogenase • Tetramer (Mr 240,000): • 2 copies of 2 different subunits • 2 Mo, 32 Fe, 30 S per tetramer • 8 dinitrogenase reductase dimers each transfers one e- to dinitrogenase

  5. Nitrogen Assimilation • Glutamine Synthetase Glutamate + NH4+ + ATP → Glutamine + ADP + Pi + H+ • Glutamate Synthase (plants, bacteria) α-Ketoglutarate + Glutamine + NADPH + H+→ 2 Glutamate + NADP+ • Net Reaction: α-Ketoglutarate + NH4+ + NADPH + ATP → Glutamate + NADP+ + ADP + Pi • Glutamate Dehydrogenase α-Ketoglutarate + NH4+ + NADPH → Glutamate + NADP+

  6. Covalent Modification Adenylylation inactivates glutamine synthase Adenylyltransferase (AT) regulated by PII Uridylylation of PII determines regulation: no UMP - adenylylation UMP – deadenylylation α-Ketoglutarate & ATP stimulate uridylylation ↑ Glutamine synthesis Glutamine & Pi inhibit uridylylation ↓ Glutamine synthesis Regulation of Glutamine Synthetase (E. coli)

  7. Transcription Activation Uridylylated PII activates gene encoding glutamine synthetase Allosteric Inhibition Glutamine synthetase is inhibited by alanine, glycine and products of glutamine metabolism Binding of each metabolite causes partial inhibition; binding of all completely inhibits enzyme Overall effect of 8 inhibitors is more than additive: example of Concerted Inhibition Regulation of Glutamine Synthetase (E. coli)

  8. Biosynthetic Reactions with Glutamine Amidotransferase reactions • Glutamine is hydrolyzed to glutamate + NH3 within the enzyme • Acceptor hydroxyl or ketone often activated with ATP Glutamine + Aspartate + ATP ↓ Glutamate + Asparagine + AMP + PPi

  9. Overview of Amino Acid Biosynthesis Ribose 5-phosphate Histidine 3-Phosphoglycerate Serine Glycine Cysteine Erythrose-4-phosphate Phosphoenolpyruvate Tryptophan Phenylalanine Tyrosine Pyruvate Alanine Valine Isoleucine Leucine Oxaloacetate Aspartate Asparagine Methionine Lysine Threonine α-Ketoglutarate Glutamate Glutamine Proline Arginine Key: nonessential conditional essential

  10. Reactions with Pyridoxal Phosphate • Transamination (aminotransferase) reactions Glutamate + Pyruvateα-Ketoglutarate + Alanine Glutamate + Oxaloacetateα-Ketoglutarate + Aspartate

  11. Amino Acids from α-Ketoglutarate

  12. α-Ketoglutarate Glutamate Glutamine Proline Glutamate g-semialdehyde Ornithine Urea Cycle Arginine Amino Acids from α-Ketoglutarate

  13. Glutamate α-Ketoglutarate ornithine aminotransferase Biosynthesis of Proline and Arginine

  14. Amino Acids from 3-Phosphoglycerate

  15. Biosynthesis of Serine and Glycine

  16. Cofactors for One-Carbon Metabolism

  17. One-Carbon Units on Tetrahydrofolate • tetrahydrofolate (H4 folate) is derived from folic acid • one-carbons on H4 folate can have 3 redox states • hydroxymethyl group of serine and formate are primary entry metabolites • reversible conversions except for production of N5-methyl H4 folate

  18. The Activated Methyl Cycle • S-Adenosyl methionine is the methyl donor for nearly all reactions • Methyl group of methionine is replenished with N5-methyl H4 folate • Methionine synthase requires coenzyme B12 (from vitamin B12)

  19. Biosynthesis of Cysteine • Only bacteria and plants can assimilate inorganic sulfur • In mammals, cysteine is conditionally essential deriving its sulfur from methionine • Cysteine is biosynthesized from serine and homocysteine, a methionine metabolite • Sulfhydryl is transferred from homocysteine to serine in two PLP dependent steps with cystathionine as intermediate

  20. Study Problem • High serum homocysteine level is a risk factor for coronary heart disease and arteriosclerosis • Some individuals with high homocysteine have variations in the cystathionine β-synthase gene • A combination of vitamin supplements is recommended for alleviating high homocysteine • Based upon the pathways for homocysteine synthesis and utilization, which 3 vitamins would you recommend?

  21. Amino Acids from Oxaloacetate and Pyruvate

  22. Amino Acids from Oxaloacetate Oxaloacetate Aspartate Asparagine Pyruvate Aspartate β-semialdehyde Dihydropicolinate Homoserine Lysine Threonine Methionine

  23. Biosynthesis of Lysine, Methione & Threonine Dihydropicolinate Lysine Methionine & Threonine

  24. Biosynthesis of Methionine

  25. Biosynthesis of Methionine

  26. cystathionine β-synthase cystathionine γ-synthase H2O H2O PLP PLP Cystathionine cystathionine γ-lyase H2O H2O cystathionine β-lyase PLP PLP Biosynthesis of Cysteine Biosynthesis of Methionine Homocysteine Serine Cysteine Homoserine γβ α-Ketobutyrate Cysteine Pyruvate Homocysteine

  27. Biosynthesis of Threonine

  28. Amino Acids from Pyruvate Threonine Alanine Pyruvate CO2 α-Ketobutyrate Pyruvate acetolactate synthetase Acetyl-CoA Same enzymes for both valine and isoleucine CO2 Isoleucine Valine Leucine

  29. Biosynthesis of Isoleucine and Valine Isoleucine Valine & Leucine

  30. Regulation of Amino Acid Biosynthesis Multiple Isozymes for • Aspartokinase (A1, A2, A3) • Homoserine dehydrogenase (B1, B2) • Threonine dehydratase (C1, C2) Allosteric regulation of selective isozymes – some unregulated Sequential feedback inhibition • Same product inhibits its biosynthetic path at multiple sites Inhibits first enzyme in pathway

  31. Biosynthesis of Aromatic Amino Acids

  32. Biosynthesis of Aromatic Amino Acids Phosphoenolpyruvate Erythrose 4-phosphate Shikimate Pyruvate Glu Gln Chorismate Anthranilate Prephenate PRPP CO2 CO2 CO2 Serine Tyrosine Phenylalanine Glyceraldehyde 3-phosphate Tryptophan

  33. Conversion of Phenylalanine to Tyrosine • Tyrosine is a conditionally essential amino acid • It can be synthesized from phenylalanine as part of its catabolic pathway

  34. Biosynthesis of Histidine PRPP ATP Glutamine Purine Biosynthesis AICAR Glutamate Histidine

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