Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum:: Role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N-5-methyl group of (6S)-methyltetrahydrofolate (CH3-H(4)folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H(4)folate and methylcob(III)amide. We have investigated binding of C-13-enriched (6R,S)-CH3-H(4)folate and (6R)-CH3-H(4)folate to MeTr by C-13 NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H(4)folate. The pH dependence of the chemical shift in the C-13 NMR spectrum for the N-5-methyl resonance indicates that MeTr decreases the acidity of the N-5 tertiary amine of CH3-H(4)folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH(3)H(4)folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H(4)folate by protonation to make the methyl group more electrophilic and the product H(4)folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-(CH3)-C-13-H(4)folate, the C-13 NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-(CH3)-C-13-H(4)folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H(4)folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T-2) of (CH3)-C-13-H(4)folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H(4)folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the C-13 resonance signal. The dissociation constant for (6R,S)-CH3-H(4)folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H(4)folate.