TRANSFORMYLASE ENZYMES DIHYDROFOLATE REDUCTASE

This proposal outlines a research effort centered on three imprtant folate requiring enzymes: two involved in de novo purine biosynthesis, the bifunctional 5'- aminoimidazole4carboxamide ribonucleotide transformylase (AICAR TFase) and the multi-functional glycineamide ribonucleotide transformylase (GAR TFase) and a third, dihydrofolate reductase. Experiments are designed to: 1) elaborate the steps and mechanism of the formyl transfer process by single turnover experiments and to find the composition of the active site by site directed irreversible inhibitors on enzymes isolated from bacterial, mammalian and avian sources; 2) delineate the requirements for inhibition of formyl transfer in normal and transformed mammalian cell lines; 3) evaluate quantitatively by a combination of site specific mutagenesis and pretransient steady state kinetics the nature of hydrophobic sidechain interactions and the identity of amino acid moieties involved in ligand binding and hydride transfer for the enzyme dihydrofolate reductase in order to establish structure/function correlations; 4) investigate spatial and kinetic relationships (total flux) between the three activities of the multifunctional protein: glycineamide ribonucleotide synthetase, glycineamide ribonucleotide transformylase and aminoimidazole ribonucleotide synthetase; 5) seek evidence for a putative avian de novo purine enzyme complex; and 6) evaluate the relative importance of de novo purine biosynthesis versus purine salvage in sustaining cellular DNA synthesis. Information gained will be used to establish the active site and mechanistic features that are common to various folate requiring enzymes (their relationship to primary structure) and the attendant implications for specific inhibition of these enzymes for chemotherapy. Quantitative dissection of the contribution of various residues to binding and catalysis for dihydrofolate reductase hopefully can be generalized to other enzymes and likewise will play a significant role in the design of site specific reagents as well as in our understanding of enzymic catalysis. Finally the data obtained should permit a rational evaluation of the importance of de novo purine biosynthesis as a drug target by providing insight into how this important biosynethic pathway functions.