Regulation of folate metabolism

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DESCRIPTION (provided by applicant): 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 studies on the catalytic mechanisms of two enzymes that catalyze the final step in methionine biosynthesis/regeneration, cobalamin-dependent methionine synthase (MetH) and cobalaminindependent methionine synthase (MetE). Both enzymes catalyze the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine. Humans do not have MetE, and so inhibitors of this essential enzyme have potential therapeutic value. Our efforts will focus on elucidating the mechanism by which methyltetrahydrofolate is activated for displacement of the methyl group, using pulse-chase and stopped flow kinetic measurements. Our studies of MetH will focus on the conformational changes required to catalyze methyl transfers between the cobalamin cofactor and the three substrates methyltetrahydrofolate, adenosylmethionine, and homocysteine. These studies will employ a combination of site-directed mutagenesis to disfavor selected conformations and spectroscopic measurements in the presence or absence of substrates to discern the effect of mutations on the spectral properties of the enzyme. The third enzyme we will study is human methylenetetrahydrofolate reductase (MTHFR), which catalyzes the formation of methyltetrahydrofolate. We have recently learned that human MTHFR is phosphorylated, and now wish to determine the significance of phosphorylation for enzyme activity, subcellular localization, and expression of active holoenzyme. MTHFR plays an important role in controlling the partitioning of one carbon units between use for nucleotide biosynthesis and incorporation into the methyl group of methionine and adenosylmethionine. We predict that phosphorylation will play an important role in modulating the flux of one-carbon units.