REGULATION OF FOLATE METABOLISM

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The long term goal of this research is to characterize the metabolic regulation of pathways of folate metabolism in both normal and diseased states, and to determine reaction mechanisms for folate dependent-enzymes. We propose to continue our studies on methylenetetrahydrofolate reductase, a flavoprotein which regulates a key branch point in folate metabolism. This enzyme catalyzes the first step in the flow of one carbon units from methylenetetrahydrofolate into the pathway for de novo biosynthesis of methyl groups, and its activity is regulated by adenosylmethionine. Rapid reaction and steady-state kinetics, coupled with photoaffinity labelling of ligand binding sites, will be used to elucidate the mode of regulation of this enzyme. We propose to purify the next enzyme in the pathway, cobalamin-dependent methionine synthase, from pig liver and to study the activation of and catalysis by enzymes from both bacterial and mammalian sources. The inhibition of these enzymes by nitrous oxide (N20), a commonly used anaesthetic agent, will also be examined. The genes coding for both the cobalamin-dependent and cobalamin-independent methionine synthase enzymes from E. coli will be cloned and sequenced and evidence for the mechanism by which the cobalamin-dependent holoenzyme represses synthesis of the cobalamin-independent enzyme will be sought. Studies are also planned to investigate the effect of ZTP, a bacterial alarmone which signals folate deprivation, on the synthesis of two key enzymes in bacterial folate biosynthesis, IMP dehydrogenase and GTP cyclohydrolase.