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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 9 Isomerizations

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

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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 9 Isomerizations

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  1. The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 9Isomerizations

  2. Isomerizations • 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

  3. [1,1]-Hydrogen ShiftRacemase with no cofactorsGlutamate racemase Not PLP - no visible absorbance Not pyruvoyl - acid hydrolysis gave no pyruvate No M2+ - EDTA has no effect No acyl intermediates - no 18O wash out of [C18O2H]Glu Not oxidation/reduction - 2H is incorporated into C-2 from 2H2O Therefore deprotonation/reprotonation mechanism

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

  5. An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase in D2O Figure 9.1

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

  7. Proposed Mechanism for Proline Racemase Scheme 9.3 Inactivation by ICH2COO- only after a reducing agent is added (RSH or NaBH4) 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. Pyridoxal 5-Phosphate (PLP) Dependent Racemases Proposed mechanism for PLP-dependent alanine racemase Scheme 9.4 Usually, a one-base mechanism

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

  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 PLP all sp2 + p atoms The -bond that is parallel to (overlapping with) the p-orbitals will break (C-H in this case)

  12. Dunathan Hypothesis for PLP Activation of the Bonds Attached to C in a PLP-Amino Acid Schiff Base 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. pyridine ring of PLP Figure 9.3 The -charge stops free rotation, which results in selective bond cleavage

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

  14. 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 solvent exchange no solvent exchange H297N mutant is capable of exchanging the -H of the S-isomer, but not the R-isomer Scheme 9.6

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

  16. Epimerases Peptide epimerases Elimination/addition (dehydration-hydration) mechanism for peptide epimerization Mechanism 1 Scheme 9.8 With 18O in the Ser OH group, no loss of 18O as H218O Therefore, mechanism 1 is unlikely.

  17. -Cleavage Mechanism for Peptide Epimerization Mechanism 2 Scheme 9.9 10 mM NH2OH has no effect on product formation Therefore, mechanism 2 is unlikely.

  18. Deprotonation/Reprotonation Mechanism Mechanism 3 Scheme 9.10 In D2O D is incorporated into product, not substrate (short incubation; monitored by electrospray ionization mass spectrometry) Deuterium isotope effect for [-2H]-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. Epimerization with Redox Catalysis 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) Scheme 9.11 two different enzymes C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0, respectively) In 2H2O 2H 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 In H218O, no incorporation of 18O into product No change in oxidation state, but is deprotonation/reprotonation reasonable?

  21. Tritium is incorporated from NAD3H into a derivative of the suspected intermediate of the UDP-glucose 4-epimerase-catalyzed reaction The enzyme requires NAD+; no exchange with solvent without OH reverse reaction proposed intermediate Scheme 9.12

  22. Proposed Mechanism for Reaction Catalyzed by UDP-Glucose 4-Epimerase Scheme 9.13 Evidence for 9.14: incubate enzyme with UDP-galactose,quench with NaB3H4. 3H at C-4 of both UDP-glucose and UDP-galactose

  23. 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 Scheme 9.14

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  42. 4-Oxalocrotonate Tautomerase Scheme 9.32 From deuterated substrates, substrate analogues, and reactions run in D2O, 9.42 to 9.44 is suprafacial (one-base mechanism)

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

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

  45. Proposed Mechanism for Isopentenyl Diphosphate Isomerase Scheme 9.35

  46. Aza-allylic Isomerization Scheme 9.36

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

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

  49. Second Half Reaction Catalyzed by Aspartate Aminotransferase Scheme 9.39 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

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