We propose to continue our investigations on the mechanism by which conversion of folate compounds to polyglutamate forms regulates one-carbon metabolism. Our investigations will proceed along several fronts: 1. Folylpolyglutamate synthetase will be purified from bacterial and mammalian sources and its substrate specificity and kinetic properties examined to determine whether they are responsible for the different types of pteroylpolyglutamates found in different cell lines. Pteroylpolyglutamate substrates and potential analog inhibitors of the synthetase will be synthesized. 2. Studies will be carried out with the Corynebacterium or E. coli folylpolyglutamate synthetase-dihydrofolate synthetase to map the folate binding site(s) of this protein and to determine whether the protein is bifunctional or whether both activities are catalyzed by a single catalytic site. 3. The metabolism of folate antagonists to polyglutamate forms will be studied in vitro using purified folypolyglutamate synthetases and in vivo using cultured mammalian cells. The ability of polyglutamate forms of these analogs to inhibit individual folate-dependent enzymes will be assessed. 4. Inhibitors of folylpolyglutamate synthetase will be 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. As pteroylmonoglutamates are poorer substrates than pteroylpolyglutamates for folate-dependent reactions, the ability of folylpolyglutamate synthetase inhibitors to increase the exogenous requirements for products of one-carbon metabolism, and to potentiate any differences in nutritional requirements between tumorigenic and nontumorigenic cells, will be studied. (B)
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