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

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

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

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

5. Tagatose 1,6-Diphosphate C 4 -epimer

6. DHAP in D 2 O in absence of G3P pro-S FDP Aldolase

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

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

9. Scheme 11.5 Evidence for Schiff Base Mechanism [ 18 O]G3P alone releases no 18 O Evidence for the involvement of an active-site lysine residue in the reaction catalyzed by fructose 1,6-diphosphate aldolase

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

11. Scheme 11.8 porphyrins (heme) corrins (vitamin B 12 )  -aminolevulinate porphobilinogen Contains M 2+ (Zn 2+ or Mg 2+) Reaction Catalyzed by Porphobilinogen Synthase [ 14 C] substrate + NaBH 4 gives [ 14 C] enzyme ICH 2 COOH and ICH 2 CONH 2 inactivate (2 different Cys) Requires a thiol reducing agent

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

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

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

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

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

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

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

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

20. Scheme 11.16 C 5 ketose C 5 aldose C 3 aldose C 7 ketose Requires thiamin diphosphate (TDP) and Mg 2+ Reaction Catalyzed by Transketolase

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

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

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

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

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

26. exchanges in D 2 O with enzyme does not exchange (but is oxidized) does not exchange and is not oxidized exchanges and is oxidized Therefore the phosphate group removes the proton from the ring (self-catalyzed) To Show Elimination Step Also Is Nonenzymatic NAD + cyclohexane - blocks ring cleavage phosphonate - not a leaving group

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

28. Scheme 11.22 retro-Claisen ICH 2 CONH 2 labels a Cys Reaction Catalyzed by Thiolase [ 14 C]acetyl-CoA gives acetylated enzyme

29. Scheme 11.23 presumably via enolate Mechanism Proposed for Thiolase

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

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

32. 4R4R Only the 4R isomer is a substrate; therefore water adds to re-face Evidence for Intermediate 11.54

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