Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase

Cobalamin-dependent methionine synthase (MetH) catalyzes the methylation of homocysteine using methyltetrahydrofolate as the methyl donor. The cobalam in cofactor serves as an intermediate carrier of the methyl. group from met hyltetrahydrofolate to homocysteine. In the two half-reactions that compris e turnover for MetH, the cobalamin is alternatively methylated by methyltet rahydrofolate and demethylated by homocysteine to form methionine. Upon bin ding to the protein, the usual dimethylbenzimidazole Ligand is replaced by the imidazole side chain of His759 [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science 266, 1669-1674]. Des pite the ligand replacement that accompanies binding of cobalamin to the ho lo-MetH protein, a MetH(2-649) fragment of methionine synthase that contain s the regions that bind homocysteine and methyltetrahydrofolate utilizes ex ogenously supplied cobalamin in methyl transfer reactions akin to those of the catalytic cycle. However, the interactions of MetH(2-649) with endogeno us cobalamin are first order in cobalamin, while the half-reactions catalyz ed by the holoenzyme are zero order in cobalamin, so rate constants for rea ctions of bound and exogenous cobalamins cannot be compared. In this paper, we investigate the catalytic rate enhancements generated by binding cobala min to MetH after dividing the protein in half and reacting MetH(2-649) wit h a second fragment, MetH(649-1227), that harbors the cobalamin cofactor. T he second-order rate constant for demethylation of methylcobalamin by Hey i s elevated BO-fold and that for methylation of cob(I)alamin is elevated 120 -fold. Thus, binding of cobalamin to MetH is essential for efficient cataly sis.