CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

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As part of an established program in pteridine biochemistry, dilhydrofolate reductase (DHFR), and dihydropteridine reductase (DHPtR), two enzymes which convert 7,8-dihydrofolate and 'quinonoid' dihydrobiopterin to their respective tetrahydro analogs, and phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine, have each been purified to homogeneity in mg quantities from rat liver; a tissue in which the three enzymes work cooperatively. In the current proposal the purified reductases are to be examined under a variety of conditions (e.g., differing pH, ionic strengths, activators, inhibitors, etc.) for cross-specificity of substrates. These results might suggest overlapping functions for the two enzymes in vivo, an observation that would be of particular interest as DHPtR is widely distributed in cell cultures and tissues which contain no ancillary hydroxylases. The dimeric, NADH requiring DHPtR (Mr approx. 51,000) will be further characterized by interaction with fluorescent probes, radioactive photolabile substrate analogs, generation of monoclonal antibodies, by preliminary high resolution NMR analysis, sequencing, and after crystallization by X-ray techniques. Wherever structural comparisons could be informative, DHFR (Mr approx. 22,500) from the same source will also be examined. A variety of fluorescent folate analogs are to be synthesised both as aids to the above enzyme characterizations and as probes for pteridine and folate cellular uptake pathways. Additionally, of long standing interest to this laboratory have been the diverse oxidation path ways of tetrahydropteridines, therefore, to complement the enzymatic investigations, a variety of methyl-substituted tetrahydropteridines have been synthesized and are to be examined in model systems to define the factors which control their paths of degradation. As a corollary to the project methotrexate will be bound to a series of synthetic peptides to examine their properties as in vivo, site-directed carriers, with improved chemotherapeutic potential.