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

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

5 10 abbreviated structure for tetrahydrofolate pteroate ring

Folic Acid (a vitamin for humans)

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

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.

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)

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

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

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

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

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

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

Transfer at Formaldehyde Oxidation State Scheme 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

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

C-5 2 H washed out in base Scheme 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

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

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

Original Proposal Scheme [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

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

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

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

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

Transfer at the Methanol Oxidation State Scheme 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)

Scheme 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

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

Scheme 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

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

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

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

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