1. The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 9 Isomerizations

2. Conversion of one molecule into another with the same formula Hydrogen shifts to the same carbon: [1,1]-H shift Hydrogen shifts to the adjacent carbon: [1,2]-H shift Hydrogen shifts to two carbon atoms away: [1,3]-H shift Isomerizations

3. Not PLP - no visible absorbance Not pyruvoyl - acid hydrolysis gave no pyruvate No M 2+ - EDTA has no effect No acyl intermediates - no 18 O wash out of [C 18 O 2 H]Glu Not oxidation/reduction - 2 H is incorporated into C-2 from 2 H 2 O Therefore deprotonation/reprotonation mechanism [1,1]-Hydrogen Shift Racemase with no cofactors Glutamate racemase

4. Scheme 9.1 [1,1]-Hydrogen Shift Amino acid racemases One base: substrate proton transferred to product (A) One-base mechanism for racemization (epimerization), (B) Two-base mechanism for racemization (epimerization) also, primary kinetic isotope effect with [2- 2 H]Glu With Glu racemase: solvent deuterium in product, not substrate Two base: incorporated proton from solvent (B)

5. Figure 9.1 in D 2 O An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase

6. Scheme 9.2 Another Test for a Two-Base Mechanism Elimination of HCl from threo-3-chloroglutamic acid by the C73A and C184A mutants for glutamate racemase

7. Scheme 9.3 Inactivation by ICH 2 COO - only after a reducing agent is added (RSH or NaBH 4 ) Proposed Mechanism for Proline Racemase Reduces active site disulfide to dithiol

8. Transition State Analogue Inhibitor Because substrates bind tightest at the transition state of the reaction, a compound resembling the TS ‡ structure would be more tightly bound TS ‡ analogue inhibitor for Pro racemase resembles 9.3

9. Scheme 9.4 Pyridoxal 5-Phosphate (PLP) Dependent Racemases Proposed mechanism for PLP-dependent alanine racemase Usually, a one-base mechanism

10. How can PLP enzymes catalyze selective bond cleavage? PLP was a coenzyme for decarboxylases (break C  -COOH bond) and now for racemases (break C  -H bond)

11. Stereochemical Relationship Between the  -Bonds Attached to C  and the p-Orbitals of the  -System in a PLP-Amino Acid Schiff Base Figure 9.2 The  -bond that is parallel to (overlapping with) the p-orbitals will break (C  -H in this case) PLP all sp 2 + p atoms

12. Figure 9.3 Dunathan Hypothesis for PLP Activation of the Bonds Attached to C  in a PLP-Amino Acid Schiff Base pyridine ring of PLP The  -charge stops free rotation, which results in selective bond cleavage The rectangles represent the plane of the pyridine ring of the PLP. The angle of viewing is that shown by the eye in Figure 9.2.

13. Scheme 9.5 No internal return in either direction Other Racemases Reaction catalyzed by mandelate racemase With (R)-mandelate no  -H exchange with solvent With (S)-mandelate there is exchange with solvent

14. Scheme 9.6 solvent exchange no solvent exchange H297N mutant is capable of exchanging the  -H of the S-isomer, but not the R-isomer A Two-base Mechanism for Mandelate Racemase that Accounts for the Deuterium Solvent Exchange Results. Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer

15. Scheme 9.7 H297N Mutant Capable of Elimination of HBr from (S)-9.5, but not from the (R)-isomer K166R mutant catalyzes elimination of HBr from the (R)-isomer, but not from the (S)-isomer Elimination of HBr from (S)-p-(bromomethyl)mandelate, catalyzed by the H297N mutant of mandelate racemase

16. Epimerases Peptide epimerases Scheme 9.8 Mechanism 1 With 18 O in the Ser OH group, no loss of 18 O as H 2 18 O Elimination/addition (dehydration-hydration) mechanism for peptide epimerization Therefore, mechanism 1 is unlikely.

17. Scheme 9.9 Mechanism 2 10 mM NH 2 OH has no effect on product formation  -Cleavage Mechanism for Peptide Epimerization Therefore, mechanism 2 is unlikely.

18. Scheme 9.10 Mechanism 3 In D 2 O D is incorporated into product, not substrate (short incubation; monitored by electrospray ionization mass spectrometry) Deprotonation/Reprotonation Mechanism Deuterium isotope effect for [  -2 H]-peptides in the L- to D-direction is different from that in the D- to L-direction (two-base mechanism) These results are consistent with mechanism 3.

19. Scheme 9.11 Epimerization with Redox Catalysis two different enzymes C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0, respectively) Proposed mechanism for dTDP-L-rhamnose synthase-catalyzed conversion of dTDP-4-keto-6-deoxy-D-glucose (9.9) to dTDP-L-rhamnose (9.10) In 2 H 2 O 2 H incorporation at both C-3 and C-5 Partial exchange gives only C-3 proton exchange, never only C-5 proton exchange (ordered sequential mechanism)

20. UDP-Glucose 4-Epimerase UDP-glucose UDP-galactose No change in oxidation state, but is deprotonation/reprotonation reasonable? In H 2 18 O, no incorporation of 18 O into product

21. Scheme 9.12 The enzyme requires NAD + ; no exchange with solvent reverse reaction without OH proposed intermediate Tritium is incorporated from NAD 3 H into a derivative of the suspected intermediate of the UDP-glucose 4-epimerase- catalyzed reaction

22. Scheme 9.13 Evidence for 9.14:incubate enzyme with UDP- galactose,quench with NaB 3 H 4. 3 H at C-4 of both UDP-glucose and UDP-galactose Proposed Mechanism for Reaction Catalyzed by UDP-Glucose 4-Epimerase

23. Scheme 9.14 Mechanism to Account for Transfer of Hydrogen from the Top Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

24. Scheme 9.15 No change in oxidation state, but NAD + required Mechanistic Pathway for the GDP- D -mannose-3,5- epimerase-catalyzed Conversion of GDP- D -mannose (9.15) to GDP- L -galactose (9.18)

25. [1,2]-H Shift Scheme 9.16 Lobry de Brun-Alberda von Ekenstein Reaction Reaction catalyzed by aldose-ketose isomerases

26. Scheme 9.17 Two Mechanisms suprafacial transfer of H cis-Enediol mechanism for aldose-ketose isomerases cis-enediol Mechanism 1

27. Scheme 9.18 Partial incorporation of solvent observed - inconsistent with hydride mechanism Hydride transfer mechanism for aldose-ketose isomerases Mechanism 2

28. Scheme 9.19 [1,3]-H Shift Enolization removes pro-R hydrogen Reaction catalyzed by phenylpyruvate tautomerase

29. Scheme 9.20 Two Conformers Possible Conformations of phenylpyruvate that would form Z- and E-enols by phenylpyruvate tautomerase

30. favored inhibitors Therefore syn geometry to E enol most likely To Test for Favored Conformation

31. Scheme 9.21 Allylic Isomerizations Carbanion mechanism for allylic isomerases This H could come from the substrate (if no solvent exchange) Mechanism 1

32. Scheme 9.22 This H comes from solvent, not from the substrate Carbocation mechanism for allylic isomerases Mechanism 2

33. Scheme 9.23 Unlikely -- [1,3]-hydride shift is allowed antarafacial, which is geometrically impossible [1,3]-Sigmatropic hydride shift mechanism for allylic isomerases Mechanism 3

34. Scheme 9.24 Carbanion Mechanism Principal reaction transfers 4  -H to 6  -position; therefore suprafacial Reaction catalyzed by 3-oxo-  5 -steroid isomerase Eliminates carbocation mechanism and [1,3] hydride shift

35. Scheme 9.26 Evidence for an Enol Intermediate in the Reaction Catalyzed by 3-Oxo-  5 -steroid Isomerase

36. Scheme 9.27 Kinetic Competence of Enol same rates Further evidence for an enol intermediate in the reaction catalyzed by 3-oxo-  5 -steroid isomerase

37. from NOE studies From Site-directed Mutagenesis, Tyr-14 is the Acid and Asp-38 the Base

38. To probe the function of Tyr-14 Scheme 9.28 Uv spectrum bound to enzyme is same as neutral amine. Structure bound to enzyme even at low pH (pK a of the phenol must be very low). Reactions Designed to Investigate the Function of Tyr-14 at the Active Site of 3-oxo-  5 -steroid Isomerase Therefore Tyr-14 does not protonate C-3 carbonyl Therefore Tyr-14 H bonds to dienolate

39. Scheme 9.29 Carbanion Mechanism Mechanism for suprafacial transfer of the 4  -proton to the 6  -proton of steroids catalyzed by 3-oxo-  5 -steroid isomerase

40. Asp-99 Located Adjacent to Tyr-14 Scheme 9.30 One mechanism for the function of Asp-99 in the active site of 3-oxo-  5 -steroid isomerase

41. equilenin Crystal structure with equilenin bound is consistent with Asp-99 and Tyr-14 both coordinated to oxyanion

42. 4-Oxalocrotonate Tautomerase Scheme 9.32 From deuterated substrates, substrate analogues, and reactions run in D 2 O, 9.42 to 9.44 is suprafacial (one-base mechanism)

43. Scheme 9.33 Carbocation Mechanism No exchange of solvent into substrate, only into product Reaction catalyzed by isopentenyl diphosphate isomerase isopentenyl diphosphatedimethylallyl diphosphate One base mechanism

44. rate is 1.8  10 -6 times K i = 14 pM Evidence for a Carbocation Mechanism transition state analogue inhibitor

45. Scheme 9.35 Proposed Mechanism for Isopentenyl Diphosphate Isomerase

46. Scheme 9.36 Aza-allylic Isomerization

47. Scheme 9.37 PLP-dependent Aminotransferase Reaction catalyzed by L-aspartate aminotransferase

48. Scheme 9.38 PMP First Half Reaction Catalyzed by Aspartate Aminotransferase

49. Scheme 9.39 Second Half Reaction Catalyzed by Aspartate Aminotransferase This is the reverse of the mechanism in Scheme 9.38

50. Crystal structures of: native enzyme with PLP bound substrate reduced onto PLP enzyme with PMP bound All are consistent with mechanisms in Schemes 8.39 and 9.38

51. pseudosubstratequinonoid form observed at 490 nm Evidence for Quinonoid Intermediate

52. Scheme 9.40  -H is transferred to the CH 2 of PMP suprafacially; therefore one-base mechanism  - 2 H removed from si-face and delivered to pro-S CH 2 of PMP Stereochemistry of Proton Transfer in the First Step Catalyzed by Many PLP-dependent Aminotransferases pro-S

53. Scheme 9.41 Cis-Trans Isomerization GSH acts as a coenzyme, not as a reducing agent No 2 H incorporated into substrate or product from 2 H 2 O Reaction catalyzed by maleylacetoacetate isomerase

54. Scheme 9.42 Proposed Mechanism for the Reaction Catalyzed by Maleylacetoacetate Isomerase

55. Scheme 9.45 Phosphate Isomerization only  -anomer binds Reaction catalyzed by phosphoglucomutases

56. Scheme 9.46 Native State of Enzyme is Phosphorylated tightly bound Shown as associative, but could be dissociative Proposed mechanism for the reaction catalyzed by phosphoglucomutases Overall retention of configuration at phosphate Double inversion

57. Scheme 9.47 Model Reaction for a Dissociative Mechanism of Phosphomutases