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 ce nter of a corrinoid/iron-sulfur protein (CFeSP), forming H(4)folate and met hylcob(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 dialysi s, fluorescence quenching, and proton uptake experiments. The results descr ibed here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., S udbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] const itute the first evidence for protonation of the pterin ring of CH3-H(4)fola te. The pH dependence of the chemical shift in the C-13 NMR spectrum for th e 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 on e proton. These results are consistent with a mechanism of activation of CH 3-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 b y cob(I)amide. When MeTr is present in excess over (6R,S)-(CH3)-C-13-H(4)fo late, 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 b ind 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 const ants for (6R)- and (6S)-CH3-H(4)folate diastereomers by fluorescence quench ing experiments. The transversal relaxation time (T-2) of (CH3)-C-13-H(4)fo late bound to MeTr is pH independent between pH 5.50 and 7.0, indicating th at 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 p H-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H(4)folate.