As part of an established program in pteridine/folate chemistry and enzymology, this proposal is focused on two areas, the comparative structure and mechanism of rat liver dihydropteridine and dihydrofolate reductases (DHPR and DHFR) and the chemistry of folate/pteridine analogs that could act as mechanistic probes or inhibitors of enzymes in folate/pteridine metabolism, and might also possess chemotherapeutic potential. The two reductases are important because they participate in metabolic cycles (purine, pyrimidine and catecholamine biosyntheses) crucial to life. DHPR has been obtained crystalline, heavy atom derivatives have been made and preliminary X-ray crystallographic measurements carried out as a prelude to obtaining the complete three dimensional structure. To help achieve this goal the enzyme has been cloned and sequenced. During the next project period it is intended to complete the X-ray work and make definitive comparisons with the known mammalian DHFR structures to relate binding sites, homology, possible evolutionary divergence and comparative reaction mechanisms. To assist in the achievement of these goals the DHPR gene is to be expressed in Escherichia coli and site specific mutations will be introduced to determine the ligand-host interactions of key amino acids at the enzyme binding sites. The information will be used to predict pterin structural analogs which might strongly inhibit DHPR, since as yet no tight binding inhibitor is known for this enzyme. Such a molecule would be invaluable for examining DHPR metabolism and possibly help discover reasons for its ubiquitous tissue distribution since its cooperative action with aromatic amino acid hydroxylases is apparent only in liver, kidney, and nerve tissue. To complement the overall structural analysis of DHPR the nucleotide binding site is to be investigated using radioactive photolabile NADH analogs, as experiments have already indicated specific uptake for such compounds. A series of novel fluorescent folate analogs are to be synthesized for use.in the above enzyme structural comparisons, these.derivatives will also be used as probes for pteridine and folate cellular transport and as markers for cellular resistance to antifolates caused by elevated DHFR. Analogs of substrates for thymidylate synthase and phosphoribosylglycinamide (GAR) formyltransferase are to be synthesi both as probes of the enzymatic mechanisms and for the determination of their cooperative chemotherapeutic potential with the established antifolate metabolites. Additionally, using variously substituted tetrahydropteridines previously synthesi in this laboratory oxidative experiments are to be carried out as models for the enzymatic aromatic amino acid hydroxylation reactions, which require reduced pterins as substrates.
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