Lecture Notes for Chapter 18 Nitrogen Metabolism

1. Essential Biochemistry Third Edition Charlotte W. Pratt | Kathleen Cornely Lecture Notes for Chapter 18 Nitrogen Metabolism

2. KEY CONCEPTS: Section 18-1 • Nitrogen fixation by the activity of nitrogenase is part of the nitrogen cycle. • Other enzymes incorporate amino groups into glutamine and glutamate. • Transaminases transfer amino groups to interconvert amino acids and α-keto acids.

3. Nitrogen fixation converts N2 into biologically useful NH4+ using nitrogenase.

4. Nitrogenase converts N2 to NH3. • Nitrogenase is a metalloprotein containing: • Fe-S clusters • Fe-Mo cofactor • FeMo cofactor • Molybdenum (green) • Iron (yellow) • Sulfur (orange) • Ammonia exists primarily in the protonated form, NH4+ .

5. Nitrogen fixation consumes a lot of ATP. • A strong reducing agent such as ferredoxin is required to donate electrons. • Net reaction is: Note that 8 electrons are required!

6. Biologically useful nitrogen also originates from nitrate. • Nitrate reductase catalyzes two reactions. • Bacteria product nitrate via nitrification • Other organisms convert nitrate back to N2 via denitrification.

7. Ammonia is assimilated into biological molecules by two proteins. • Glutamine synthetase • Produces glutamine from glutamate • Glutamate synthase • Produces glutamate from glutamine • Nomenclature • Synthetase uses ATP • Synthase does not use ATP Let’s look at each reaction next…

8. Glutamate Synthetase Reaction • Glu is phosphorylated. • Ammonium ion displaces inorganic phosphate.

9. Glutamate Synthase Reaction • Nitrogen is assimilated into a citric acid cycle intermediate to produce Glu. • Gln is deaminated to produce a second Glu.

10. Net Reaction of Nitrogen Fixation Glutamine Synthetase Glu + ATP + NH4+ Gln + ADP + Pi α-ketoglutarate + Gln + NADPH + H+Glu + Glu + NADP+ Glutamate Synthase + H+

11. Structure of E. coli Glu Synthetase • 12 identical subunits • Changes in activity at one active site can be communicated to other active sites. • Activity is tightly regulated to maintain a supply of accessible amino groups.

12. Transamination moves amino groups between compounds. • Transaminase = aminotransferase • A transaminase catalyzes the transfer of an amino group to an a-keto acid. • Transamination is reversible.

13. Transaminases use pyridoxal-5’-phosphate as a cofactor. • PLP is derived from the essential nutrient, pyridoxine (vitamin B6).

14. Transaminase Binding to PLP • Amino group transfer occurs through a Lys. • Schiff base formation is key in the mechanism. Let’s look at the mechanism more closely…

15. The Mechanism of Transamination

16. The Mechanism of Transamination

17. The Mechanism of Transamination

18. The Mechanism of Transamination

19. The Mechanism of Transamination

20. The Mechanism of Transamination

21. The Mechanism of Transamination

22. KEY CONCEPTS: Section 18-2 • Alanine, arginine, asparagine, aspartate, glutamate, glutamine, glycine, proline, and serine are synthesized from the intermediates of glycolysis and the citric acid cycle. • Bacteria and plants synthesize amino acids with sulfur (cysteine and methionine), branched chains (isoleucine, leucine, and valine), and aromatic groups (phenylalanine, tryptophan, and tyrosine) as well as histidine, lysine, and threonine. • Glutamate and tyrosine are modified to generate neurotransmitters and hormones.

23. Nitrogen Metabolism in Context • Amino acids are synthesized from intermediates of glycolysis and the citric acid cycle. • Nonessential amino acids can be synthesized. • Essential amino acids must be obtained from food.

25. Several amino acids are easily synthesized from common intermediates.

26. Several amino acids are easily synthesized from common intermediates.

27. Several amino acids are easily synthesized from common intermediates.

28. Several amino acids are easily synthesized from common intermediates.

29. Tetrahydrofolate functions as a carrier of 1-C units in several reactions of amino acid and nucleotide metabolism.

30. In the conversion of Ser to Gly, methylenetetrahydrofolate is produced.

31. Amino acids with sulfur, branched chains, or aromatic groups are more difficult to synthesize.

32. Amino acids with sulfur, branched chains, or aromatic groups are more difficult to synthesize.

33. Amino acids with sulfur, branched chains, or aromatic groups are more difficult to synthesize.

34. In plants and bacteria, synthesis of aromatic amino acids involves formation of chorismate. Not made by animals

35. Tyr is produced from Phe via hydroxylation.

36. Tryptophan synthase catalyzes the final two steps of Trp synthesis. • Movement of a reactant between two active sites is called channeling.

37. Tryptophan Synthase

38. Histidine is not formed from carbohydrate metabolites.

39. Amino acids are the precursors of some signaling molecules. • Abbreviated as GABA • Neurotransmitter • Glutamate derivative

40. Catecholamines are derived from amino acids.

41. Catecholamines are derived from amino acids.

42. Serotonin is the precursor of melatonin.

43. KEY CONCEPTS: Section 18-3 • AMP and GMP are derived from the purine nucleotide IMP. • Pyrimidine nucleotide synthesis produces UTP and then CTP. • Ribonucleotide reductase converts NDPs to dNDPs using a free radical mechanism. • dUMP is methylated to produce dTMP. • Nucleotides are degraded for excretion and to supply materials for salvage or other pathways.

44. Purine nucleotide synthesis yields IMP and then AMP and GMP.

45. AMP and GMP Synthesisfrom IMP

46. Pyrimidine nucleotide synthesis yields UTP. UMP is phosphorylated to UDP and then UTP

47. CTP is produced from UTP.

48. Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides.

49. Thymidine nucleotides are produced by methylation.

50. Methotrexate is an effective anticancer drug. • In cancer cells, enzymes involved in nucleotide synthesis are highly active. • Methotrexate is a competitive inhibitor of dihydrofolate reductase.