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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 11 Aldol and Claisen Reactions and Retroreactions

The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 11 Aldol and Claisen Reactions and Retroreactions. Aldol Reactions Generalized aldol reaction. aldehyde or ketone. -hydroxyaldehyde or -ketone. Scheme 11.1.

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The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 11 Aldol and Claisen Reactions and Retroreactions

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  1. The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 11Aldol and Claisen Reactions and Retroreactions

  2. Aldol ReactionsGeneralized aldol reaction aldehyde or ketone -hydroxyaldehyde or -ketone Scheme 11.1

  3. AldolasesGeneral mechanisms for type I (pathway a) and type II (pathway b) aldolases Type I Schiff base mechanism Type II Metal ion catalyzed Scheme 11.2

  4. Reaction Catalyzed by Fructose 1,6-Diphosphate Aldolase Scheme 11.3 dihydroxyacetone phosphate (DHAP) glyceraldehyde 3- phosphate (G3P) fructose 1,6-phosphate (FDP) 11.3 + 11.4 (3S,4S)11.5 (3S,4R) tagatose 1,6-diphosphate aldolase

  5. Tagatose 1,6-Diphosphate C4-epimer

  6. FDP Aldolase DHAP in D2O in absence of G3P pro-S

  7. Ordered Product Release Rate of incorporation of 14C into fructose 1,6-diphosphate (FDP) from [14C]glyceraldehyde 3-phospate (G3P) and [14C]dihydroxyacetone phosphate (DHAP) catalyzed by fructose 1,6-diphosphate aldolase Figure 11.1 Therefore in the reverse reaction G3P is released faster than DHAP

  8. Ordered Reaction of Fructose 1,6-Diphosphate Aldolase Scheme 11.4 DHAP is released last in retroreaction; therefore it binds first in forward reaction (ordered reaction) (Principle of Microscopic Reversibility)

  9. Evidence for Schiff Base Mechanism Evidence for the involvement of an active-site lysine residue in the reaction catalyzed by fructose 1,6-diphosphate aldolase Scheme 11.5 [18O]G3P alone releases no18O

  10. Overall Proposed Mechanism and Stereochemistry for Fructose 1,6-Diphosphate Aldolase Scheme 11.6 pro-S si

  11. Reaction Catalyzed by Porphobilinogen Synthase porphyrins (heme) Scheme 11.8 corrins (vitamin B12) porphobilinogen -aminolevulinate Contains M2+ (Zn2+ or Mg2+) Requires a thiol reducing agent ICH2COOH and ICH2CONH2 inactivate (2 different Cys) [14C] substrate + NaBH4 gives [14C] enzyme

  12. Proposed Mechanism (imine, then aldol) for the Reaction Catalyzed by Porphobilinogen Synthase Scheme 11.9

  13. Alternative Proposed Mechanism (aldol, then imine) for the Reaction Catalyzed by Porphobilinogen Synthase Scheme 11.10

  14. Retro-aldol Reactions Reaction catalyzed by ATP citrate-lyase retro-aldol oxaloacetate citrate Scheme 11.11 Isotope exchange and kinetics support a double displacement mechanism with a phosphoryl enzyme intermediate

  15. Reasonable Intermediates can substitute for citrate and ATP gives product in absence of citrate, ATP, CoASH

  16. Maybe more than two intermediates Exchange between [14C]ADP and ATP in the absence of all other substrates --- enzyme phosphorylation ATP phosphorylation of ATP citrate-lyase and exchange with [14C]ADP Scheme 11.12

  17. Phosphoryl Enzyme Isolated with [32P]ATP ATP phosphorylation of ATP citrate-lyase Scheme 11.13

  18. Mechanism Proposed for the Reaction Catalyzed by ATP Citrate-lyase may not form Scheme 11.14

  19. Possible Alternative Mechanism Proposed for the Reaction Catalyzed by ATP Citrate-lyase Scheme 11.15 Whenever there are at least two steps in a mechanism, consider reversing two of the consecutive steps

  20. Reaction Catalyzed by Transketolase Scheme 11.16 C3 aldose C7 ketose C5 aldose C5 ketose Requires thiamin diphosphate (TDP) and Mg2+

  21. Mechanism Proposed for Transketolase equivalent Scheme 11.17 TDP provides a way of stabilizing a carbanion at the carbonyl

  22. Transketolase in Organic Synthesis Synthesis of substituted D-threo-trihydroxylated ketones catalyzed by transketolase Scheme 11.18

  23. Reaction Catalyzed by Dehydroquinate Synthase Scheme 11.19 Substrate and product are in the same oxidation state Requires NAD+

  24. Originally Proposed Mechanism for Dehydroquinate Synthase double bond activates elimination This mechanism suggests the enzyme is capable of catalyzing 5 different reactions! Scheme 11.20

  25. Model Study to Show that the Last 2 Steps are Really Nonenzymatic isolated

  26. To Show Elimination Step Also Is Nonenzymatic cyclohexane - blocks ring cleavage phosphonate - not a leaving group NAD+ does not exchange (but is oxidized) exchanges in D2O with enzyme does not exchange and is not oxidized exchanges and is oxidized Therefore the phosphate group removes the proton from the ring (self-catalyzed)

  27. Claisen Reactions Generalized Claisen reaction Scheme 11.21 -H of thioesters is 100 times more acidic than that of esters C-C bond forms with inversion of stereochemistry (aldol reactions go with retention)

  28. Reaction Catalyzed by Thiolase retro-Claisen Scheme 11.22 ICH2CONH2 labels a Cys [14C]acetyl-CoA gives acetylated enzyme

  29. Mechanism Proposed for Thiolase presumably via enolate Scheme 11.23

  30. Reaction Catalyzed by Kynureninase retro-Claisen aldol Scheme 11.24

  31. Mechanism Proposed for Kynureninase re face addition (behind) or PhCHO Scheme 11.25

  32. Evidence for Intermediate 11.54 4R Only the 4R isomer is a substrate; therefore water adds to re-face

  33. TS‡ Analogue Based on 11.59 Ki = 70 nM (300-fold lower than Km of substrate)

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