The mechanism of conversion of folate compounds to polyglutamate forms, and how this process regulates one-carbon metabolism and anti-folate cytotoxicity, will be investigated. Several approaches will be used. 1. The catalytic and kinetic mechanisms, and the substrate binding sites, of human folylpolyglutamate synthetase (FPGS) will be characterized. Candidate active site residues will be identified by affinity labeling and their involvement in binding or catalysis confirmed by site-directed mutagenesis. The function of identified residues will be ascertained by steady state and rapid reaction kinetic analyses. Crystallization of the protein will be attempted to aid in the analysis of how substrates and inhibitors bind to the protein. 2. The human folypolyglutamate synthetase gene will be characterized and the regulation of folypolyglutamate synthetase expression under different growth and nutrient conditions will be studied. The mechanism by which antifolates modulate folypolyglutamate synthetase activity and how this process affects cytotoxicity will be investigated. The mechanism by which mitochondrial and cytosolic folypolyglutamate synthetase isozymes are generated will be ascertained. 3. Regulatory aspects of folate and anti-folate metabolism in mammalian cells and the role of changes in folypolyglutamate synthetase levels in the cytotoxic effects of anti-folate agents will be investigated. The cell systems used will include MCF-7, a human breast cancer cell line, and Chinese hamster ovary cell transfectants expressing various levels of human folypolyglutamate synthetase activity in the mitochondria and cytosol. The long term goals of these studies are (1) to understand the basic mechanisms underlying the regulation of anti-folate, folate and one carbon metabolism and to develop models explaining these important physiological processes and (2) to use the information obtained in protein chemistry studies to design new anti-folate agents which are inhibitors of folypolyglutamate synthetase and to design 'pro-drugs', anti-folates that are inhibitors of cell growth when metabolized to polyglutamate forms, with improved activities for this enzyme.
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