REGULATION OF FOLATE METABOLISM

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The long term goals of this research are to elucidate the catalytic mechanisms of enzymes that use tetrahydrofolate derivatives as cofactors, and to study the regulation of one carbon metabolism. This research will emphasize mechanistic studies of two enzymes, cobalamin-dependent methionine synthase and serine hydroxymethyltransferase, that are potential targets for cancer chemotherapy, but for which no specific inhibitors are currently available. Strategies are proposed for the design of specific active site-directed inhibitors of each of these enzymes. Cobalamin- dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate (CH3-H4folate) to homocysteine, with the enzyme- bound cobalamin prosthetic group serving as an intermediary in the methyl transfer. We plan to determine the mode by which the substrate, CH3- H4folate, is activated for methyl transfer by using UV-visible and NMR spectroscopy to determine whether the substrate is protonated or oxidized in dead-end ternary complexes. We will investigate the mechanism by which the enzyme is inactivated by the commonly used anesthetic, nitrous oxide, and will attempt to design agents that are capable of inactivation of the enzyme by a similar mechanism. Related studies on the activation of methionine synthase by reductive methylation, and on the catalytic mechanism of a cobalamin-independent methionine synthase that catalyzes a closely similar reaction are also proposed. We will continue our studies on the catalytic mechanism of serine hydroxymethyltransferase, a pyridoxal phosphate-dependent enzyme that catalyzes the transfer of the beta-carbon of serine to H4folate to yield glycine and CH2-H4folate. These studies will attempt to elucidate the role of H4folate in the reaction mechanism and will explore the use of halogenated amino acids as potential inhibitors of catalysis. The availability of highly specific inhibitors for both methionine synthase and serine hydroxymethyltransferase will not only suggest approaches for chemotherapeutic targeting of these enzymes, but will also permit an assessment of the role of these enzymes in cellular metabolism.