Derivatives of Amino Acids and Metabolism of Nucleotides

1. Derivatives of Amino Acids and Metabolism of Nucleotides CH353 January 29, 2008

2. Anabolic Role of the Citric Acid Cycle Error: glycine not glutamate provides carbons for purines Purines X Biosynthesis of amino acids & derivatives from citric acid cycle intermediates require anaplerotic reactions (red arrows) for replenishing metabolites

3. Derivatives of Amino Acids • Porphyrins and Heme • Glycine + Succinyl-CoA (animals) • Glutamate (bacteria & plants) • Non-ribosomal peptide synthesis • peptidoglycan, antibiotics • glutathione (glutamate + cysteine + glycine) • Modified amino acids • plant compounds, neurotransmitters, polyamines • Nucleotide heterocyclic bases • purines and pyrimidines

4. Biosynthesis of Heme animals heme precursor bacteria, plants

5. Biosynthesis of Heme

6. Genetic Deficiencies in Heme Biosynthesis

7. Catabolism of Heme purple Regulated step: 3 isozymes green yellow Important serum antioxidant Bile pigment yellow (oxidized) red-brown (reduced)

8. Reactions with Monooxygenases • Use 2 reductants for O2 (mixed-function oxygenases) • One reductant accepts an O atom • Other reductant provides 2 H’s to the second O atom • General Reaction: AH + BH2 + O–O → A–OH + B + H2O

9. Biosynthesis of Nitric Oxide • NO involved in intercellular signaling • NO synthase (a mixed-function oxygenase) • dimer, similar to NADPH cytochrome P450 reductase • cofactors: FMN, FAD, tetrahydrobiopterin, Fe3+ heme • catalyzes a 5 e- oxidation

10. Biosynthesis of Creatine • metabolite for storage of high energy transfer potential phosphate • phosphorylated at high [ATP] • amidinotransferase exchanges amino acids • glycine for ornithine • 1 substrate and 1 product same as for arginase reaction except different amidino group acceptor • glycine instead of water • S-adenosylmethionine methyl donor

11. Biosynthesis of Glutathione • reducing agent (redox buffer) • non-ribosomal peptide synthesis • carboxyl groups activated with ATP (acyl phosphate intermediates)

12. Non-ribosomal Peptide Synthesis • Microbial peptides are synthesized by multi-modular synthases; similar to fatty acid biosynthesis • Modular complexes of enzymes for recognition, activation, modification and condensation of a specific amino acid to the growing polymer • Features use of unusual amino acids, D-enantiomers, and non-α peptide bonds • Peptidoglycans, antibiotics and ionophores

13. Reactions with Pyridoxal Phosphate • Amino acid racemase reactions L-alanine ↔ D-alanine Inhibitors of alanine racemase: Antibiotics – peptidoglycan biosynthesis

14. Biosynthesis of Plant Compounds • phenylalanine, tyrosine, tryptophan precursors for plant compounds: • lignin (phenolic polymer) • indole-3-acetate (auxin) • tannins • alkaloids, e.g. morphine • flavors, e.g. cinnamon, nutmeg, cloves, vanilla, cayenne pepper

15. Reactions with Pyridoxal Phosphate • Decarboxylase reactions Histidine → Histamine + CO2 Ornithine → Putrescine + CO2

16. Biosynthesis of Neurotransmitters Pathways involve decarboxylases and mixed-function oxygenases (monooxygenases)

17. Biosynthesis of Spermidine and Spermine Pathway involves decarboxylases and S-adenosylmethione alkylation

18. African Sleeping Sickness • Caused by Trypanosoma brucei rhodesiense • Vaccines are ineffective: repeated change of coat antigen • Therapy based on inhibitor of polyamine biosynthesis

19. Mechanism of Ornithine Decarboxylase

20. Inhibition of Ornithine Decarboxylase Ornithine DMF-Ornithine

21. Study Problem • Antihistamines are compounds that block histamine synthesis or binding to its receptor • Histamine is synthesized from histidine by a pyridoxal phosphate dependent decarboxylase • Design an antihistamine drug candidate, based upon the mechanism for decarboxylation • Show the structure and its proposed mechanism of action

22. Overview of Nucleotide Metabolism • Nucleotide functions • Activated precursors for synthesis of RNA, DNA and cofactors • Activation of biosynthetic precursors • Energy for cellular processes • Signal transduction • Biosynthetic pathways • de novo synthesis of purines and pyrimidines • differ in order of attachment of ribose to base • salvage pathways • reacting a base with activated 5-phosphoribose (PRPP)

23. Precursors for Nucleotide Biosynthesis • 5-phosphoribosyl-1-pyrophosphate ribose phosphate pyrophosphokinase Ribose 5-phosphate + ATP → 5-phosphoribosyl-1-pyrophosphate + AMP

24. Precursors for Nucleotide Biosynthesis Tetrahydrofolate (H4 folate) derivatives • N5,N10-methylene-H4 folate • thymidylate biosynthesis • N5-formyl-H4 folate • purine biosynthesis

25. Precursors for Nucleotide Biosynthesis • Amino Acids • Glycine for purine biosynthesis • Aspartate for pyrimidine biosynthesis • Amino Acid Nitrogen • α-amino group of aspartate (purines) aspartate + [acceptor] + ATP → succinyl-amino-[acceptor] + ADP+ Pi succinyl-amino-[acceptor] → amino-[acceptor] + fumarate • amide group of glutamine (purines, pyrimidines) glutamine + [acceptor] + ATP → amino-[acceptor] + glutamate + ADP+ Pi

26. O O O R–C–OPO3–2 R–C–NHR’ R–C–O– OPO3–2 NHR’ C–C–R C–C–R C–C–R O H Activation of Amino Acceptors • carboxylate or carbonyl acceptor are activated with ATP • acyl-phosphate or phospho-enol intermediates formed • nucleophilic substitution of phosphate with amino group R’NH2 PO4–2 ATP ADP

27. 5 2 6 3 7 4 1 Biosynthesis of the Purine Ring • Multi-step synthesis from many precursors • (numbers indicate order of addition to purine ring from PRPP)

28. Purine Biosynthesis • glutamine-PRPP amidotransferase • glutamine donates amide nitrogen to activated 5-phosphoribose (PRPP) • committed step for purine synthesis • product unstable t½ = 30 seconds • GAR synthetase • glycine carboxyl activated with ATP • Pi displaced; amide bond formed • GAR transformylase • N10-formyl tetrahydrofolate donates formyl group to glycine amino group • FGAR amidotransferase • ATP activates carbonyl group • amidotransfer displaces Pi

29. Purine Biosynthesis • FGAM cyclase (AIR synthetase) • ATP activates carbonyl • cyclization of imidazole ring in bacteria & fungi: • N5-CAIR synthetase • ATP activates HCO3- • carbamoylation of exocyclic amine • N5-CAIR mutase • transfer of carboxylate to ring in higher eukaryotes: • AIR carboxylase • formation of only C-C bond • no cofactors or ATP required

30. Purine Biosynthesis • SAICAR synthetase • aspartate is amino donor • ATP activates carboxylate • aspartate amino replaces Pi • SAICAR lyase • fumarate is eliminated • steps 8 & 9 analogous to urea cycle • AICAR from histidine biosynthesis • AICAR transformylase • N10-formyl H4 folate donates formyl group to glutamine-derived amine • IMP synthase • cyclization of second purine ring • ATP activation not required

31. Organization of Purine Biosynthetic Enzymes • Purine biosynthesis organized into multienzyme complexes • In eukaryotes, multifunctional proteins for: • Steps 1, 3 & 5 • Steps 6a & 8 • Steps 10 & 11 • In bacteria, separate enzymes associate in large complexes • Channeling of intermediates avoids dilution of reactants

32. Synthesis of Adenylate and Guanylate • AMP synthesis uses GTP for activation; amine from aspartate • GMP synthesis uses ATP for activation; amide from glutamine Reciprocal Regulation: • GTP for needed for AMP synthesis • ATP needed for GMP synthesis

33. Regulation of Purine Biosynthesis in E. coli Feedback Inhibition (negative) • Inhibition of 1st step in common pathway by IMP, AMP & GMP • Inhibition of 1st step in branch • AMP inhibits AMP synthesis • GMP inhibits GMP synthesis • Inhibition of PRPP synthesis by phosphorylated end products ADP, GDP and others Reciprocal Regulation (positive) • Requirements of: • ATP for GMP synthesis • GTP for AMP synthesis

34. Purine Biosynthesis Hypoxanthine (a purine) is assembled on the ribose 5-phosphate → Inosinate (IMP) Precursors: PRPP Glycine H4 folate-formate (2) HCO3– Glutamine (amide-N) (2) Aspartate (amino-N) IMP → AMP IMP → XMP → GMP Pyrimidine Biosynthesis Orotate (a pyrimidine) is made first then added to ribose 5-phosphate → Orotidylate Precursors: Carbamoyl phosphate HCO3– Glutamine (amide-N) Aspartate PRPP Orotidylate → UMP → UDP→ UTP → CTP Nucleotide Biosynthesis

35. Pyrimidine Biosynthesis Carbamoyl Phosphate Synthetase II • cytosolic CPS II enzyme involved in pyrimidine biosynthesis • mitochondrial CPS I involved in arginine & urea synthesis • bacteria have single enzyme for both functions Steps: • bicarbonate phosphate synthesis (1st activation) • carbamate synthesis (NH3 from glutamine hydrolysis) • carbamoyl phosphate synthesis (2nd activation)

36. Carbamoyl Phosphate Synthetase Bacterial enzyme has 2 subunits (blue & grey) with 3 active sites joined by a substrate channel (yellow wire mesh) • 1st site: Glutamine releases NH4+ (glutamine in green) • 2nd site: HCO3– is phosphorylated with ATP and reacts with NH4+ to form carbamate (ADP in blue) • 3rd site: Carbamoyl phosphate is synthesized by phosphorylating carbamate with ATP (ADP in red)

37. Pyrimidine Biosynthesis • aspartate transcarbamoylase • activated carbamoyl group transferred to amine group of aspartate • Pi displaced; amide bond formed • committed step in pyrimidine synthesis • dihydroorotase • cyclization of pyrimidine ring • dihydroorotate dehydrogenase • oxidation of C-C bond using NAD+ • orotate phosphoribosyl transferase • pyrimidine ring (orotate) is transferred to activated 5-phosphoribose (PRPP) • PPi lost; aminoglycan bond formed • analogous to pyrimidine salvage

38. Pyrimidine Biosynthesis • orotidylate decarboxylase • catalyzes synthesis of UMP • very efficient enzyme • uridylate kinase • nucleoside monophosphate kinase specific for UMP • nucleoside diphosphate kinase • generic enzyme for (d)NDP’s • cytidylate synthetase • an amidotransferase • UTP is aminated using glutamine • carbonyl group is activated with ATP to form acyl phosphate intermediate Cytidine 5’-triphosphate (CTP)

39. Pyrimidine Biosynthesis Enzyme Complexes • Eukaryotes have a multifunctional protein with the first 3 enzymes in pyrimidine biosynthetic pathway C carbamoyl phosphate synthetase II A aspartate transcarbamoylase D dihydroorotase • CAD has 3 identical polypeptides (Mr 230,000) each with sites for all 3 reactions

40. Regulation of Pyrimidine Biosynthesis • Feedback inhibition of 1st step aspartate transcarbamoylase (ATCase) by CTP • Bacterial ATCase has: • 6 catalytic subunits • 6 regulatory subunits • Allosteric inhibition: • 2 conformations of ATCase: active ↔ inactive • binding of CTP to regulatory subunits shifts conformation active → inactive • ATP reverses effect of CTP

41. Activation of Nucleotides • Nucleoside monophosphate kinases • specific enzyme for each base (e.g. adenylate kinase) • nonspecific for ribose (ribose or 2’-deoxyribose) ATP + NMP ADP + NDP • Nucleoside diphosphate kinase • generic enzyme, nonspecific for base or ribose • nonspecific for phosphate donor or acceptor NTP + NDP NDP + NTP donor acceptor acceptor donor

42. Nucleotides for DNA Synthesis 2 Modifications: • ribonucleotides reduced to 2’-deoxyribonucleotides NDP → dNDP • uracil (uridylate) methylated to thymine (thymidylate) dUMP → dTMP

43. Reduction of Nucleotides • NDP is reduced to dNDP by reduced form of ribonucleotide reductase • Oxidized form of ribonucleotide reductase is reduced by either glutaredoxin or thioredoxin • Oxidized form of glutaredoxin is reduced by glutathione • Oxidized form of thioredoxin is reduced by FADH2 • Oxidized glutathione and FAD are reduced by NADPH

44. Regulation of Ribonucleotide Reductase Ribonucleotide Reductase (E. coli) • Active sites are between each R1 and R2 subunit • Two R2 subunits each contain a tyrosyl radical and a binuclear Fe3+ cofactor • Two R1 subunits each have sites for enzyme activity and substrate specificity • The (d)NTP bound to substrate specificity sites determines which NDP is reduced to dNDP

45. Binding at activity regulatory sites: ATP activates enzyme dATP inhibits enzyme Binding at substrate specificity sites: ATP or dATP: ↑dCDP ↑dUDP dTTP: ↑dGDP↓dCDP ↓dUDP dGTP: ↑dADP↓dGDP ↓dCDP ↓dUDP Regulation of Ribonucleotide Reductase

46. CTP nucleoside diphosphate kinase cytidylate synthetase UTP uridylate kinase UMP Biosynthesis of Thymidylate • Precursors for thymidylate (dTMP) synthesis may arise from (d)CTP or (d)UTP pools

47. Cyclic pathway for conversion of dUMP to dTTP • Thymidylate synthase uses N5,N10-Methylene-H4 folate as both one-carbon source and reducing agent • Dihydrofolate reductase reduces H2 folate → H4 folate with NADPH • Serine hydroxymethyl transferase reaction restores N5,N10-Methylene-H4 folate • Net reaction: dUMP + NADPH + serine → dTMP + NADP+ + glycine

48. Inhibitors of glutamine amidotransferases: Block purine & pyrimidine biosynthesis Inhibitors of thymidylate synthesis: thymidylate synthase dihydrofolate reductase Chemotherapeutic Agents

49. Chemotherapy Targets

50. Group Study Problem • Conversion of dUTP to dTTP by thymidylate synthase requires N5,N10-Methylene-H4 folate as both one-carbon source and reducing agent • N5,N10-Methylene-H4 folate and glycine are produced in a reversible reaction whereby the hydroxymethyl group of serine in transferred to H4 folate • What effect may an elevated glycine:serine ratio during photorespiration have on DNA synthesis? January 31, 2008