The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 8 Decarboxylation

1. The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 8Decarboxylation

2. Decarboxylation ReactionsDriving force for decarboxylation Scheme 8.1

3. Decarboxylation of -Keto Acids Scheme 8.2 Decarboxylation is accelerated in acid

4. Cyclic Transition State for Decarboxylation of -Keto Acids Scheme 8.3 Strong acids are needed to protonate carbonyls (pKa -7)

5. Amine-catalyzed decarboxylation of -keto acids Protonation of Imines (pKa about +7) is Easy Scheme 8.4

6. Reaction Catalyzed by Acetoacetate Decarboxylase Scheme 8.5

7. Schiff Base Mechanism Fate of the ketone oxygen in the reaction catalyzed by acetoacetate decarboxylase Suggests a Schiff base mechanism Scheme 8.6 In D2O, D is incorporated into acetone pKa of Lys-115 is 5.9; adjacent to Lys-116, which lowers pKa by about 4.5 pKa units Why?

8. Reaction of the K115C Mutant of Acetoacetate Decarboxylase with 2-Bromoethylamine inactive mutant Scheme 8.7 after aminoethylation it is active Lys-116 mutants are still active (but less than WT) pKa of Lys-115 in Lys-116 mutants is >9 Aminoethylation of K116C - lowers pKa of K115 back to 5.9

9. Schiff Base Mechanism Proposed mechanism for acetoacetate decarboxylase Scheme 8.8

10. Test for Schiff Base Mechanism NaBH4 reduction during the reaction catalyzed by acetoacetate decarboxylase isolated Scheme 8.9

11. Metal Ion-catalyzed Mechanism Alternative to Schiff base mechanism Scheme 8.10 no loss of carbonyl oxygen With 14C substrate + NaBH4 no 14C protein

12. Proposed Mechanism for the Decarboxylation of (S)--acetolacetate (8.11) Catalyzed by -Acetolactate Decarboxylase inversion of stereochemistry Scheme 8.11

13. -Hydroxy Acids Proposed mechanism for the isocitrate dehydrogenase-catalyzed conversion of isocitrate (8.17) to -ketoglutarate (8.19) oxalosuccinate isocitrate -ketoglutarate Scheme 8.15 Oxalosuccinate is not detected. Also no partial reactions, but it is presumably formed

14. Not All Decarboxylations Need Schiff Base or M2+ Reaction catalyzed by phosphogluconate dehydrogenase Scheme 8.16 6-phosphogluconate ribulose 5-phosphate Experiments?

15. Proposed Mechanism for the Reaction Catalyzed by Phosphogluconate Dehydrogenase Scheme 8.17

16. -Keto Acids Improbable decarboxylation of -keto acids Scheme 8.18

17. Cofactor Required Diphosphorylation of thiamin Scheme 8.19 thiamin (vitamin B1) thiamin diphosphate coenzyme vitamin

18. Abbreviated Form for TDP

19. Resonance Stabilization of Thiazolium Ylide Scheme 8.20 exchangeable in neutral D2O at room temperature pKa estimated 13-18

20. Proposed Mechanism for Autodeprotonation of C-2 of Thiamin Diphosphate Without N-1 N or N-4 NH2 it is not active Without N-3 N it is active Scheme 8.21

21. Stable, but C-2 proton not very acidic Ideal heterocycle 100 times more acidic, but does not catalyze -keto acid decarboxylation and is easily hydrolyzed at pH 7

22. Mechanism of Thiamin Diphosphate-dependent Enzymes Nonoxidative decarboxylation of -keto acids: (A) the reaction catalyzed by pyruvate decarboxylase, (B) the reaction catalyzed by acetolactate synthase pyruvate decarboxylase acetolactate synthase Scheme 8.26 acetoin

23. Benzoin CondensationChemical model for formation of 8.45 Scheme 8.27

24. Mechanism for the Benzoin Condensation nucleophile electrophile Scheme 8.29 catalyst

25. Proposed Mechanism for the Reaction Catalyzed by Acetolactate Synthase like -CN electrophile nucleophile Scheme 8.30 catalyst

26. Examples of Oxidative Decarboxylation of -Keto Acids -KG succinyl-CoA Scheme 8.33 Multienzyme complexes 5 different coenzymes involved

27. -Keto Acid Dehydrogenase Proposed mechanism for the reaction catalyzed by dihydrolipoyl transacetylase dihydrolipoyl transacetylase pyruvate decarboxylase lipoic acid TDP -CO2 FAD lipoic acid NAD+ acetyl lipoamide reduced lipoic acid Scheme 8.34 dihydrolipoyl dehydrogenase

28. Coenzyme A

29. Alternative Proposed Mechanism for the Reaction Catalyzed by Dihydrolipoyl Transacetylase Scheme 8.35

30. Proposed Mechanism for the Reaction Catalyzed by Dihydrolipoyl Dehydrogenase dihydrolipoyl dehydrogenase Scheme 8.37

31. Amino Acid Decarboxylation covalently bound via Schiff base to a Lys residue Conversion of pyridoxine to pyridoxal 5-phosphate (PLP) pyridoxine (vitamin B6) Pyridoxal 5-phosphate (PLP) coenzyme Scheme 8.38

32. The First Step Catalyzed by All PLP-dependent Enzymes, the Formation of the Schiff Base between the Amino Acid Substrate and PLP Scheme 8.39

33. Schiff Base Formation Increases the Electrophilicity of the Carbonyl Reaction of an amine with an imine Reaction of an amine with an aldehyde 30x faster than Scheme 8.40

34. Reaction Catalyzed by PLP Decarboxylases (different enzymes for different amino acids) neutralizes acidic conditions increases intracellular pressure Scheme 8.41

35. To Provide Evidence for a Schiff Base with an Active Site Lysine Residue Reduction and hydrolysis of PLP enzymes. Scheme 8.42

36. If Substrate Is Added before NaBH4

37. Incorrect hydrolytic mechanism for PLP-dependent enzymes No 18O from H218O Found in CO2 Scheme 8.43

38. Proposed Mechanism for PLP-dependent Decarboxylases stereospecific incorporation of proton electron sink to stabilize anion Scheme 8.44

39. Pyruvoyl-Dependent Decarboxylases - Identification and Differentiation Proposed mechanism for pyruvoyl-dependent decarboxylases (amino acids) Scheme 8.45 PLP and PQQ enzymes have absorbance >300 nm Pyruvoyl enzymes - no absorbance >300 nm PLP and PQQ enzymes do not give the products shown above Differences

40. Inactivation of S-Adenosylmethionine Decarboxylase by its Substrate inactivation S-adenosylmethionine (SAM) Scheme 8.46

41. Biosynthesis of the Active-site Pyruvoyl Group of Histidine Decarboxylase prohistidine decarboxylase both 18O end up here Pathway b is valid only if the hydroxide released is the same one that hydrolyzes the amide bond. Scheme 8.47

42. Other Decarboxylations Proposed addition/elimination for orotidine 5-monophosphate decarboxylase Scheme 8.48

43. Proposed Zwitterion Mechanism for Orotidine 5-Monophosphate Decarboxylase Scheme 8.49

44. Model Study for the First Mechanism Model reactions for the addition/elimination mechanism for orotidine 5-monophosphate decarboxylase Scheme 8.50

45. Support for the Second Mechanism(actually, disproof of the first mechanism) incubate with enzyme - no rehybridization by 13C NMR no secondary deuterium isotope effect

46. More Evidence Against the First Mechanism X = Br, Cl inhibitors X = F substrate excellent substrate no rehybridization

47. When R = COOH OMP decarboxylase is the most proficient enzyme known kcat = 39 s-1 knon = 2.8  10-16s-1 (nonenzymatic) kcat/knon = 1.4  1017 (rate enhancement)

48. Rationale for Direct Decarboxylation original proposal protonation at O-2 protonation at O-4 based on calculations Two atoms of Zn2+ in active site - may stabilize negative charge

49. Reaction Catalyzed by Mevalonate Diphosphate Decarboxylase Decarboxylative elimination Scheme 8.51

50. Both are poor substrates Destabilize a carbocation intermediate