1. The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 8 Decarboxylation

2. Scheme 8.1 Decarboxylation Reactions Driving force for decarboxylation

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

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

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

6. Scheme 8.5 Reaction Catalyzed by Acetoacetate Decarboxylase

7. Scheme 8.6 Schiff Base Mechanism Suggests a Schiff base mechanism In D 2 O, D is incorporated into acetone pK a of Lys-115 is 5.9; adjacent to Lys-116, which lowers pK a by about 4.5 pK a units Fate of the ketone oxygen in the reaction catalyzed by acetoacetate decarboxylase Why?

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

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

10. Scheme 8.9 Test for Schiff Base Mechanism NaBH 4 reduction during the reaction catalyzed by acetoacetate decarboxylase isolated

11. Scheme 8.10 Metal Ion-catalyzed Mechanism Alternative to Schiff base mechanism With 14 C substrate + NaBH 4 no loss of carbonyl oxygen no 14 C protein

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

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

14. Scheme 8.16 Not All Decarboxylations Need Schiff Base or M 2+ 6-phosphogluconate Experiments? ribulose 5-phosphate Reaction catalyzed by phosphogluconate dehydrogenase

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

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

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

18. Abbreviated Form for TDP

19. Scheme 8.20 exchangeable in neutral D 2 O at room temperature pK a estimated 13-18 Resonance Stabilization of Thiazolium Ylide

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

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

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

23. Scheme 8.27 Benzoin Condensation Chemical model for formation of 8.45

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

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

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

27. Scheme 8.34  -Keto Acid Dehydrogenase pyruvate decarboxylase dihydrolipoyl transacetylase lipoic acid TDP reduced lipoic acid acetyl lipoamide Proposed mechanism for the reaction catalyzed by dihydrolipoyl transacetylase -CO 2 dihydrolipoyl dehydrogenase FAD NAD + lipoic acid

28. Coenzyme A

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

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

31. Scheme 8.38 Amino Acid Decarboxylation covalently bound via Schiff base to a Lys residue pyridoxine (vitamin B 6 ) Pyridoxal 5-phosphate (PLP) coenzyme Conversion of pyridoxine to pyridoxal 5-phosphate (PLP)

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

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

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

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

36. If Substrate Is Added before NaBH 4

37. Scheme 8.43 No 18 O from H 2 18 O Found in CO 2 Incorrect hydrolytic mechanism for PLP-dependent enzymes

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

39. 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 Proposed mechanism for pyruvoyl-dependent decarboxylases (amino acids) Pyruvoyl-Dependent Decarboxylases - Identification and Differentiation Differences

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

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

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

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

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

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

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

47. When R = COOH OMP decarboxylase is the most proficient enzyme known k cat = 39 s -1 k non = 2.8  10 -16 s -1 (nonenzymatic) k cat /k non = 1.4  10 17 (rate enhancement)

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

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

50. Both are poor substrates Destabilize a carbocation intermediate

51. Very Potent Inhibitor - TS ‡ Analogue Inhibitor mimics a carbocation

52. Scheme 8.52 Proposed Carbocation Mechanism for Mevalonate Diphosphate Decarboxylase