The mechanisms by which conversion of folate compounds to polyglutamate forms regulates one-carbon metabolism will be investigated. Several approaches will be used. 1. Studies will be carried out with the E. coli folylpolyglutamate synthetase-dihydrofolate synthetase to map the folate binding site(s), to determine whether the protein is bifunctional, and to investigate the catalytic mechanism. Site specific mutagenesis, affinity labeling, and kinetic and fluorimetric analyses will be used to address these questions. These studies will aid in the logical design of inhibitors of this enzyme. 2. Mammalian folylpolyglutamate synthetase will be characterized with emphasis on its specificity for polyglutamate substrates whose metabolism is rate limiting for folate and antifolate retention by cells. 3. Regulatory aspects of one carbon and antifolate metabolism in mammalian cells and the role of polyglutamate formation in the functioning of metabolic cycles of one carbon metabolism and the cytotoxic action of antifolates will be investigated. Model systems used will include hamster cell transfectants expressing the E. coli or human folylpolyglutamate synthetase gene, and containing modified folylpolyglutamate distributions and folylpolyglutamate synthetase levels, and an in vitro model using folylpolyglutamate synthetase. 4. Inhibitors of folylpolyglutamate synthetase will be synthesized and tested for their ability to cause cellular folate depletion by preventing the formation of pteroylpolyglutamates, the forms of the vitamin that are retained by tissues. Inhibitors of folylpolyglutamate synthetase, by virtue of their potential ability to produce a functional folate deficiency in tissues, should increase the nutritional requirement for folate and/or products of one-carbon metabolism by mammalian cells and may exacerbate any differences in nutritional requirements between normal and tumorigenic cells.
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