Protonation state of methyltetrahydrofolate in a binary complex with cobalamin-dependent methionine synthase

N5 -Methyltetrahydrofolate (CH3 -H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent meth ionine synthase (MetH, EC 2.1.1.3). Nucleophilic displacement of a methyl g roup attached to a tertiary amine is a reaction without an obvious preceden t in bioorganic chemistry. Activation of CH3-H(4)folate by protonation prio r to transfer of the methyl group has been the favored mechanism. Protonati on at N5 would lead to formation of an aminium cation, and quaternary amine s such as 5,5-dimethyltetrahydropterin have been shown to transfer methyl g roups to cob(I)alamin. Because CH3-H(4)folate is an enamine, protonation co uld occur either at N5 to form an aminium cation or on a conjugated carbon with formation of an iminium cation. We used C-13 distortionless enhancemen t by polarization transfer (DEPT) NMR spectroscopy to infer that CH3-H4fola te in aqueous solution protonates at N5, not on carbon. CH3-H(4)folate must eventually protonate at N5 to form the product H(4)folate; however, this p rotonation could occur either upon formation of the binary enzyme-CH3-H-4-f olate complex or later in the reaction mechanism. Protonation at N5 is acco mpanied by substantial changes in the visible absorbance spectrum of CH3-H( 4)folate. We have measured the spectral changes associated with binding of CH3-H4folate to a catalytically competent fragment of MetH over the pH rang e from 5.5 to 8.5. These studies indicate that CH3-H4folate is bound in the unprotonated form throughout this pH range and that protonated CH3-H(4)fol ate does not bind to the enzyme. Our observations are rationalized by seque nce homologies between the folate-binding region of MetH and dihydropteroat e synthase, which suggest that the pterin ring is bound in the hydrophobic core of an alpha (8)beta (8) barrel in both enzymes. The results from these studies are difficult to reconcile with an S(N)2 mechanism for methyl tran sfer and suggest that the presence of the cobalamin cofactor is important f or CH3-H(4)folate activation. We propose that protonation of N5 occurs afte r carbon-nitrogen bond cleavage, and we invoke a mechanism involving oxidat ive addition of Co1+ to the N5-methyl bond to rationalize our results.