One carbon units bound to tetrahydrofolate are utilized for the de novo biosynthesis of purines and thymidylate and for the provision of methyl groups for the biological methylation reactions that involve adenosylmethionine as the methyl donor. The long term goals of this research are to study the catalytic mechanisms of enzymes that use tetrahydrofolate derivatives as cofactors, and to study the regulation of one carbon metabolism. Because the availability of one carbon units is one of the factors that limits the rate of growth of cells, these studies are relevant to the design of chemotherapeutic inhibitors of folate-dependent enzymes. They are also relevant to our understanding of the factors that control the level of plasma homocysteine, an independent risk factor for the development of cardiovascular disease. The proposed studies focus on three folate-dependent enzymes: human methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate in a reaction which commits one-carbon units to provision of methyl groups for adenosylmethionine-dependent methylations, and cobalamin-dependent and cobalamin-independent methionine synthases from Escherichia coli. Methionine synthases catalyze methyl transfer from methyltetrahydrofolate to homocysteine, to produce tetrahydrofolate and methionine. Based on research with MTHFR from E. coli, a model has been developed in which enzyme activity is regulated by the availability of folate derivatives; this model will be tested for its applicability to the regulation of the human MTHFR. Studies of the catalytic mechanisms of cobalamin-dependent and cobalamin-independent methionine synthases from Escherichia coli are also proposed. These studies will focus on the mechanism of activation of the substrate, methyltetrahydrofolate, for transfer of the methyl group and on the catalytic role of the essential zinc ions in these two enzymes, and will employ a wide variety of kinetic, spectroscopic, and stereochemical techniques.
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