1. The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 7 Carboxylations

2. Carboxylations General Concepts A carbanion (or carbanionic character) must be generated where carboxylation is to occur. Metal ion complexation of the oxygen atom of the keto and enol forms can increase the acidity of an adjacent C-H bond by 4-6 orders of magnitude CO 2 is an excellent electrophile for carboxylation, but at physiological pH, it is in low concentration Predominant form is bicarbonate (HCO 3 - ), which is actually a nucleophile To convert bicarbonate into an electrophile, it must be activated either by phosphorylation or dehydration Must be a stabilized carbanion.

3. In general, all enzymes utilize CO 2 except for phosphoenolpyruvate carboxylase and the biotin- dependent enzymes, which use bicarbonate To determine which is the substrate: Put CO 2 into the enzyme reaction at a concentration approximating its K m value, and incubate with sufficient enzyme so that a significant amount of product is produced in the first few seconds. There are two possible outcomes (Figure 7.1, next slide):

4. Figure 7.1 Carboxylations electrophile CO 2 + H 2 O H 2 CO 3 nucleophile (equilibrium ~ 1 min) Possible outcomes when CO 2 is added to a carboxylase Also, repeat in the presence of carbonic anhydrase (catalyzes hydrolysis of CO 2  H 2 CO 3 ) Test for whether CO 2 or HCO 3 - is the substrate for a carboxylate

5. Scheme 7.1 CO 2 as Carboxylating Agent PEP oxaloacetate If run in H 2 18 O with CO 2, no 18 O in products Reaction catalyzed by PEP carboxykinase Addition of [ 14 C]pyruvate does not give [ 14 C]oxaloacetate. Pyruvate or enolpyruvate are not free intermediates. (need large amount of enzyme so no nonenzymatic conversion of CO 2 to HCO 3 - )

6. Scheme 7.2 In the absence of CO 2, the enzyme acts like a kinase (H + in place of CO 2 ) pyruvate PEP carboxykinase-catalyzed reaction of PEP with ADP (no CO 2 )

7. Scheme 7.3 If the carboxylase reaction is run in D 2 O in the presence of malate DH/NADH, no D is in the malate; oxaloacetate Reduction of Oxaloacetate by Malate Dehydrogenase malate Malate dehydrogenase traps oxaloacetate to prevent nonenzymatic enolization. therefore no enol of oxaloacetate formed.

8. Scheme 7.4 This mechanism is excluded by the previous result: Hypothetical Mechanism for PEP Carboxykinase that Involves the Enolate of Oxaloacetate

9. Scheme 7.5 Running the reaction in reverse inversion of stereochemistry Stereochemistry of the Reaction Catalyzed by PEP Carboxykinase Excludes covalent catalytic mechanism

10. Scheme 7.6 This Mechanism is Excluded: Inconsistent with a double-inversion mechanism for PEP carboxykinase

11. Scheme 7.7 Possible Mechanism for PEP Carboxykinase Concerted mechanism for PEP carboxykinase (or stepwise without release of intermediates)

12. Scheme 7.8 Same as PEP carboxykinase except P i instead of nucleotide diphosphate All mechanistic experiments are the same for the two enzymes Reaction Catalyzed by Phosphoenolpyruvate Carboxytransphosphorylase (oxaloacetate)

13. Figure 7.2 re Alkene stereochemistry nomenclature rules for (Z)-1-bromo-1-propene (7.8) Stereochemical Rules Needed to Determine Stereochemistry of PEP Carboxytransphosphorylase

14. Figure 7.3 si re Alkene Nomenclature Rules for (E)-1- bromo-1-propene (7.9) si-re or re-si? Cite the side with the highest priority group (in this case, Br) Front face is named re-si face

15. Scheme 7.10 (Z)-[3- 3 H]PEP With (E)-[3- 3 H]PEP, 98% 3 H in fumarate; therefore carboxylation from si-re face anti- elimination observed 98% loss as 3 H 2 O Two Possible Stereochemical Outcomes for Carboxylation of PEP Catalyzed by PEP Carboxytransphosphorylase P-O bond of PEP breaks, but C-O bond of PEP breaks with EPSP synthase fumarate

16. Scheme 7.11 blood-clotting proteins binds Ca 2+ Vitamin K Cycle for Carboxylation of Proteins

17. Figure 7.4 -proteases Holds the proteases to the appropriate cells, triggering the blood-clotting cascade Calcium-dependent Binding of Clotting Proteins to Cell Surfaces

18. Scheme 7.12 erythro- and threo- erythro- F - elimination, but not threo-; Test for Carbanion vs. Radical Mechanisms for Vitamin K Carboxylase therefore stereospecific (carbanion)

19. Scheme 7.13 carboxylation with inversion of stereochemistry Stereochemical Outcome of Vitamin K Carboxylase-catalyzed Carboxylation of (2S,4R-fluoroglutamate)

20. Scheme 7.14 But where does vitamin K fit into the mechanism? Proposed Vitamin K Carboxylase-catalyzed Carboxylation of Glutamate Residues via a Carbanionic Intermediate

21. Model Study for Function of Vitamin K Not a strong enough base to deprotonate 7.20 Dieckmann condensation Reaction does not work in absence of O 2 Scheme 7.15 strong base Base Strength Amplification Mechanism Chemical model study for the activation of vitamin K 1 as a base Model for reduced vitamin K

22. Scheme 7.16 (not 1 O 2 ) When run in 18 O 2, 0.95 mol atom 18 O in epoxide 0.17 mol atom 18 O in quinone oxygen Two Proposed Mechanisms for Activation of Vitamin K 1 as a Base

23. To Determine Which Ketone is Involved Incubation in 16 O 2 atmosphere gives loss of 0.17 mol atom 18 O from 7.23, none from 7.24 Therefore, the ketone next to the methyl group is involved in the reaction

24. Scheme 7.18 To account for much loss of 18 O from substrate Modified Base Strength Amplification Mechanism for Vitamin K Carboxylase

25. Scheme 7.19 Bicarbonate as the Carboxylating Agent PEP No H 2 18 O formed (high enzyme concentration, short time at alkaline pH) Reaction catalyzed by PEP carboxylase Therefore HCO 3 -, not CO 2

26. Scheme 7.20 Note: nucleophilic mechanisms concerted stepwise associative stepwise dissociative No partial exchange detected ([ 14 C]pyruvate does not give [ 14 C]PEP) Concerted (A), Stepwise Associative (B), and Stepwise Dissociative (C) Mechanisms for PEP Carboxylase Therefore, either concerted or intermediate not released

27. Evidence for Stepwise Mechanism in H 2 18 O inversion concerted is suprafacial sigmatropic; therefore retention Also, rate is independent of pH, but the carbon isotope effect for H 13 CO 3 - decreases with increasing pH. Not possible with concerted Evidence for dissociative mechanism: Using methyl PEP and HC 18 O 3 - more than 1 18 O in P i and substrate recovered has 18 O in nonbridging position of phosphate; therefore reversible CO 2 + P i formed (see next slide)

28. Note: the ultimate carboxylating agent is CO 2 Scheme not in text (after Scheme 7.20) Mechanism for Incorporation of 18 O into Substrate Non-bridging 18 O C

29. Biotin-dependent Enzymes Multisubunit enzymes Enzyme reactions with HC 18 O 3 - give P i with one 18 O and product with 2 18 O atoms (bicarbonate) Scheme 7.24 Covalent attachment of d-biotin to an active site lysine residue

30. Figure 7.5 Diagnostic method for biotin - add avidin K D = 1.3  10 -15 M Reactions Catalyzed by Biotin-dependent Carboxylases

31. Scheme 7.25 Mechanism of Biotin-Dependent Carboxylases No substrate or product needed Suggests ATP activates bicarbonate Partial exchange reaction of 32 P i into ATP (in absence of substrate) with biotin-dependent carboxylases

32. Scheme 7.26 Mechanism for Partial Exchange of 32 P i into ATP with Biotin-dependent Carboxylases

33. Scheme 7.27 Partial Exchange Reaction of [ 14 C]ADP into ATP with Biotin-dependent Carboxylases

34. Scheme 7.28 [ 14 C]product substrate, HCO 3 - ATP, M 2+ [ 14 C]substrate Mechanism for Partial Exchange Reaction of [ 14 C]ADP into ATP with Biotin- dependent Carboxylases (reaction is reversible)

35. Scheme 7.29 Evidence for Enzyme-Bound Intermediate In the absence of pyruvate get a carboxylated enzyme if pyruvate is added Carboxylated enzyme is unstable to acid (pH 4.5), but stable to base (0.033 N KOH) [ 14 C] carboxylated enzyme in base purified by gel filtration then stabilized by CH 2 N 2 treatment (makes methyl ester) Pyruvate carboxylase-catalyzed incorporation of 14 C from H 14 CO 3 - into the enzyme

36. Scheme 7.30 Isolated; X-ray crystal structure The X in previous Scheme Isolation of N 1 -methoxycarbonylbiotin from the Reaction Catalyzed by Pyruvate Carboxylase Followed by Diazomethane Trapping of the N-carboxybiotin

37. Figure 7.6 1. 2. 3. Six Possible Mechanisms for Formation of N 1 -carboxybiotin

38. Figure 7.6 4. 5. 6. In the presence of HCO 3 - but absence of biotin, biotin carboxylase catalyzes hydrolysis of ATP; with HC 18 O 3 - one 18 O incorporated into P i ; therefore supports formation of carboxyphosphate (mechanism 1).

39. Scheme 7.31 carboxyphosphate Mechanism for the Formation of Carboxyphosphate in the Reaction Catalyzed by Acetyl-CoA Carboxylase

40. Figure 7.8 Initial evidence for concerted: retention of configuration at  -carbon Possible Mechanisms for Transfer of CO 2 from N 1 -carboxybiotin to Substrates

41. Scheme 7.37 Evidence for Stepwise Mechanism Double isotope fractionation test: Compare with If concerted, should show both 2 H and 13 C isotope effects (C-H bond broken and C-C bond made simultaneously) If stepwise, not necessarily so Also, if stepwise, 13 C isotope effect could be different with and without 2 H 13 (V/K) for 13 CH 3 COCOOH 1.0227 13 (V/K) for 13 CD 3 COCOOH 1.0141 (calculated value is 1.0136) therefore stepwise Transcarboxylase and propionyl-CoA carboxylase- catalyzed elimination of HF from  -fluoropropionyl-CoA