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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation

The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation. Redox Without a Coenzyme. Internal redox reaction. Reaction Catalyzed by Glyoxalase. Scheme 3.1. methylglyoxal. lactic acid. Looks like a Cannizzaro reaction. Cannizzaro Reaction Mechanism. Scheme 3.2.

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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation

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  1. The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 3Reduction and Oxidation

  2. Redox Without a Coenzyme Internal redox reaction

  3. Reaction Catalyzed by Glyoxalase Scheme 3.1 methylglyoxal lactic acid Looks like a Cannizzaro reaction

  4. Cannizzaro Reaction Mechanism Scheme 3.2

  5. Reactions Catalyzed by Glyoxalase I and Glyoxalase II reduced oxidized glutathione Scheme 3.3

  6. Glutathione (GSH)

  7. Hydride Mechanism for Glyoxalase oxidized reduced Scheme 3.4 Intramolecular Cannizzaro reaction

  8. Evidence for a hydride mechanism - when run in 3H2O, lactate contains less than 4% tritium • NMR experiment provided evidence for a proton transfer mechanism: Enzyme reaction followed by NMR • At 25 °C in 2H2O, 15% deuterium was incorporated • At 35 °C, 22% deuterium was incorporated

  9. Enediol Mechanism for Glyoxalase cis-enediol Scheme 3.5

  10. Reaction of Glyoxalase with Fluoromethylglyoxal Another test for the mechanism Scheme 3.6 same oxidation state

  11. Hydride Mechanism for the Reaction of Glyoxalase with Fluoromethylglyoxal Scheme 3.7

  12. Enediol Mechanism for the Reaction of Glyoxalase with Fluoromethylglyoxal Scheme 3.8

  13. Hydride Mechanism for the Reaction of Glyoxalase with Deuterated Fluoromethylglyoxal Scheme 3.9 F- loss decreased deuterium isotope effect

  14. Enediol Mechanism for the Reaction of Glyoxalase with Deuterated Fluoromethylglyoxal deuterium isotope effect Scheme 3.10 F- loss increased

  15. increased F- loss supports enediol mechanism

  16. Redox Reactions that Require CoenzymesNicotinamide Coenzymes (Pyridine Nucleotides) • Pyridine nucleotide coenzymes include nicotinamide adenine dinucleotide (NAD+, 3.10a), nicotinamide adenine dinucleotide phosphate (NADP+, 3.10b), and reduced nicotinamide adenine dinucleotide phosphate (NADPH, 3.11b)

  17. NAD(P)+ NAD(P)H Enzyme without coenzyme bound - apoenzyme Enzyme with coenzyme bound - holoenzyme coenzyme apoenzyme holoenzyme Called reconstitution

  18. Abbreviated Forms NAD(P)H (reduced) NAD(P)+ (oxidized)

  19. Coenzymes typically derived from vitamins (compounds essential to our health, but not biosynthesized) • Pyridine nucleotide coenzymes derived from nicotinic acid (vitamin B3, also known as niacin)

  20. Biosynthesis of Nicotinamide Adenine Dinucleotide (NAD+) nicotinic acid (vitamin B3) niacin from ATP Scheme 3.11

  21. Reactions Catalyzed by Pyridine Nucleotide-containing Enzymes Oxidation potential NAD+/NADH is -0.32 V Figure 3.1

  22. Reactions Catalyzed by Alcohol Dehydrogenases Mechanism Scheme 3.12 In 3H2O, no3H in NAD(P)H Hydride mechanism

  23. Reaction Catalyzed by Alcohol Dehydrogenases Using Labeled Alcohol Scheme 3.13 No *H found in H2O Supports hydride mechanism

  24. Cyclopropylcarbinyl Radical Rearrangement Test for a radical intermediate Scheme 3.14

  25. Test for the Formation of a Radical Intermediate with Lactate Dehydrogenase Scheme 3.15 No ring cleavage - evidence against radical mechanism

  26. Chemical Model for the Potential Formation of a Cyclopropylcarbinyl Radical during the Lactate Dehydrogenase-catalyzed Reaction Scheme 3.16 Should have seen ring opening in the enzyme reaction if a cyclopropylcarbinyl radical formed

  27. Nonenzymatic Reduction of -Chloroacetophenone Another test for a radical intermediate Nonenzymatic reaction radical reduction product Scheme 3.18

  28. Horse Liver Alcohol Dehydrogenase-Catalyzed Reduction of -Haloacetophenones Scheme 3.19 hydride reduction product (stereospecific) X = F, Cl, Br Supports no radical intermediate When X = I, get mixture of 3.25 (X = I) + Electron transfer is possible if the reduction potential is low enough (radical reduction product)

  29. Stereochemistry An atom is prochiral if by changing one of its substituents, it changes from achiral to chiral

  30. Stereochemistry:Determination of the chirality of an isomer of alanine R,S Nomenclature Figure 3.2

  31. Determination of Prochirality Figure 3.3

  32. Determination of sp2 Carbon Chirality • Determine the priorities of the three substituents attached to the sp2 carbon according to the R,S rules • If the priority sequence is clockwise looking down from top, then the top is the re face; if it is counterclockwise, then it is the si face

  33. Determination of Carbonyl and Alkene (sp2) Chirality Figure 3.4

  34. Reaction of Yeast Alcohol Dehydrogenase (YADH) with (A) [1,1-2H2]ethanol and NAD+ and (B) Ethanol and [4-2H]NAD+ Scheme 3.20

  35. Reaction of YADH with (A) [4-2H]NAD2H Prepared in Scheme 3.20A; (B) Reaction of YADH with [4-2H]NAD2H Prepared in Scheme 3.20B; (C) Reaction of YADH with 3.28 and NAD+ No 2H stereospecific No H Scheme 3.21

  36. only one H is transferred re-face

  37. Not all enzymes transfer the same hydride (A) Reaction of YADH with [1,1-2H2]ethanol and NAD+; (B) Reaction of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) with the cofactor produced in A and glycerate 1,3-diphosphate pro-R pro-S transferred Scheme 3.22

  38. Anti- and syn- conformations of NADH Transition State for Hydride Transfer Figure 3.5 syn-axial electrons assist Boat-like TS‡

  39. The enzyme may drive equilibrium Boat-boat equilibria of NADH Figure 3.6

  40. Oxidation of Amino Acids to Keto Acids Possible mechanism for the reaction catalyzed by glutamate dehydrogenase Hydride transfer Scheme 3.24

  41. Oxidation of Aldehydes to Carboxylic Acids (A) Covalent catalytic mechanism for the oxidation of aldehydes by aldehyde dehydrogenases; (B) noncovalent catalytic mechanism for the oxidation of aldehydes by aldehyde dehydrogenases covalent catalysis Hydride transfers Scheme 3.25 via hydrate

  42. Oxidation of Deoxypurines to Purines Mechanism for the oxidation of inosine 5-monophosphate by inosine 5-monophosphate dehydrogenase inosine MP xanthine MP Scheme 3.27

  43. An Atypical Use of NAD+Reaction catalyzed by urocanaseNAD+ in a Nonredox Reaction Scheme 3.28

  44. Urocanase Reaction Run with a [13C] Pseudo-substrate apo-urocanase reconstituted with [13C]NAD+ “substrate” exchangeable proton

  45. Adduct Isolated after Chemical Oxidation NMR determined

  46. Mechanism Proposed for Urocanase solvent incorporated exchangeable Scheme 3.29

  47. Biosynthetic conversion of riboflavin to FMN and FAD Flavin Coenzymes riboflavin (vitamin B2) FMN FAD Scheme 3.31

  48. Interconversion of the Three Oxidation States of Flavins oxidized semiquinone reduced some covalently attached to The protein at these positions Scheme 3.32

  49. Redox Reactions Catalyzed by Flavin-dependent Enzymes Figure 3.8

  50. Oxidases vs. Dehydrogenases Mechanisms for an oxidase-catalyzed oxidation of reduced flavin to oxidized flavin Scheme 3.33 only if spin inversion occurs Oxidases use O2 for reoxidation of reduced flavin coenzyme

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