1. The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 12 Formylations, Hydroxymethylations, and Methylations

2. Transfer of one-carbon units can be oligomer of up to 12 Glu residues tetrahydrofolate named as polyglutamate derivatives of tetrahydrofolate (H 4 PteGlu n ) [Pte - pteroate] Tetrahydrofolate-dependent Enzymes

3. 5 10 abbreviated structure for tetrahydrofolate pteroate ring

4. Folic Acid (a vitamin for humans)

5. Reduction of Folate to Tetrahydrofolate Scheme 12.1 dihydrofolate Reactions catalyzed by dihydrofolate reductase (DHFR)

6. Scheme 12.2  -cleavage pro-S hydrogen added/removed (retention of configuration) Ordered mechanism: no conversion of Ser to H 2 C=O unless tetrahydrofolate is bound Generation of the Transferring Carbon Unit Serine hydroxymethyltransferase-catalyzed formation of formaldehyde via a proposed  -cleavage mechanism. The asterisk indicates the carbon unit that becomes the one-carbon unit transferred in tetrahydrofolate-dependent enzymes.

7. The one-carbon unit can be transferred in 3 oxidation states Scheme 12.3 N 5 -methylene H 4 Pte N 5,N 10 -methylene H 4 Pte N 10 -methylene H 4 Pte K eq = 3.2 x 10 4 in favor of 12.7 Transfer of One-Carbon Units Serine hydroxymethyltransferase-catalyzed reaction of formaldehyde and tetrahydrofolate to methylenetetrahydrofolate Transfer at the formaldehyde oxidation state (transfer HOCH 2 - group)

8. Scheme 12.5 N 5,N 10 -methenyltetrahydrofolate cyclohydrolase N 10 -formyl H 4 Pte N 5 -formyl H 4 Pte Transfer at the Formate Oxidation State (transfer formyl group) Oxidation of N 5,N 10 -methylenetetrahydrofolate to N 5,N 10 - methenyltetrahydrofolate catalyzed by methylenetetrahydrofolate dehydrogenase and hydrolysis of N 5,N 10 -methenyltetrahydrofolate to N 5,N 10 -methenyltetrahydrofolate cyclohydrolase

9. N 5 -methyl H 4 Pte Requires NADPH and FAD to make 12.13 from N 5 -methylene H 4 Pte Transfer at the Methanol Oxidation State (transfer methyl group)

10. Excludes [1,3]-hydride shift Excludes tautomerization of N 5 -methylene H 4 Pte to 12.14 Reaction Run Backwards with [6- 3 H]-12.13 Releases No 3 H and Does Not Transfer 3 H to Methyl

11. Scheme 12.7 Proposed Mechanism for the Reduction of N 5,N 10 -methylenetetrahydrofolate by N 5,N 10 -methylenetetrahydrofolate Reductase

12. Scheme 12.8 With [5- 3 H]-deazaFADH 2, 3 H transferred to methyl group, consistent with this mechanism Proposed Alternative Hydride Mechanism for N 5,N 10 -Methylenetetrahydrofolate Reductase

13. Proposed mechanism for glycinamide ribonucleotide (GAR) transformylase Scheme 12.9 GARFGAR Transfer of a Formyl Group Third step in biosynthesis of purines

14. Transfer at Formaldehyde Oxidation State Scheme 12.10 exchanges reduced (normally CH 2 OH) oxidized C-5 H exchanges with solvent Reaction catalyzed by thymidylate synthase (an anomalous transfer of a methylene group) Last step in de novo biosynthesis of thymidylate inverse 2° isotope effect  rehybridization of C-5 from sp 2  sp 3 inverse 2° isotope effect at C-6 also

15. Scheme 12.11 transferred to Transfer of the C-6 Hydrogen of N 5,N 10 - Methylenetetrahydrofolate to the Methyl Group of Thymidylate Catalyzed by Thymidylate Synthase

16. C-5 2 H washed out in base Scheme 12.12 note: C-5 and C-6 are rehybridized to sp 3 Thiols are more effective than hydroxide Chemical Model Study for Thymidylate Synthase-catalyzed Exchange of the C-5 Hydrogen of 2-Deoxyuridine-5-monophosphate

17. Scheme 12.13 structure identified by X-ray Inactivation of Thymidylate Synthase by 5-Fluoro-2-deoxyuridylate

18. Scheme 12.14 Proposed Mechanism for the First Part of the Reaction Catalyzed by Thymidylate Synthase Based on Inactivation Complex with 5-Fluoro-2-deoxyuridylate

19. Original Proposal Scheme 12.15 [1,3]-H shift suprafacial Not allowed by Woodward-Hoffman rules Should have occurred with 5-F analogue, but does not Highly unlikely [1,3]-H shift mechanism for reduction of the substrate catalyzed by thymidylate synthase

20. To Rationalize Stability of 5-F Adduct Scheme 12.16 Proposed mechanism for the second part of the reaction catalyzed by thymidylate synthase when F, it is stable

21. Precedence for Elimination Mechanism Scheme 12.17 Model study for the formation of the C-5 exo- methylene intermediate proposed in the reaction catalyzed by thymidylate synthase

22. Enzymatic Intermediate Trapped with  -Mercaptoethanol Scheme 12.18 isolated Trapping of the proposed C-5 exo-methylene intermediate during catalytic turnover of thymidylate synthase

23. Scheme 12.19 Alternative proposed electron transfer mechanism for the reduction of the exo-methylene intermediate in the reaction catalyzed by thymidylate synthase Alternative to Hydride Transfer

24. Transfer at the Methanol Oxidation State Scheme 12.20 Reaction catalyzed by the cobalamin-independent methionine synthase Two different forms of methionine synthase: one transfers CH 3 directly from N 5 -methyl H 4 PteGlu one first transfers CH 3 to a cobalt complex (cobalamin)

25. Scheme 12.21 SN2SN2 increases leaving group ability (model for protonated N 5 -Me-H 4 PteGlu) Enzyme requires Zn 2+ (coordinates to the thiol S) Model Study for the Reaction Catalyzed by the Cobalamin-independent Methionine Synthase

26. corrin ring methylcobalamin from the methylation of cob(I)alamin by N 5 -MeH 4 PteGlu Cobalamin-dependent Methionine Synthase

27. Scheme 12.22 retention of Me configuration Cleland notation (A) Reaction Catalyzed by Cobalamin-dependent Methionine Synthase (B) Cleland Diagram for the Reaction Catalyzed by Cobalamin-dependent Methionine Synthase Enzyme reaction pathway

28. Scheme 12.23 Model Study for the Methylation of Cob(I)alamin during the Reaction Catalyzed by the Cobalamin-dependent Methionine Synthase

29. S-Adenosylmethionine (SAM)-Dependent Transfer of CH 3 Scheme 12.24 rare attack at C-5 ATP SAM more common methylating agent Proposed mechanism for the synthesis of S-adenosylmethionine catalyzed by methionine adenosyltransferase

30. Scheme 12.25 With chiral CH 3 group gives inversion of stereochemistry Generalized Reaction Catalyzed by S-adenosylmethionine-dependent Methyltransferases SN2SN2

31. Scheme 12.26 indolylpyruvate indolmycin inversion of stereochemistry Stereochemistry of Methylation of Indolylpyruvate in the Biosynthesis of Indolmycin