This proposal seeks support for a study of the mechanism of several enzymes involved in the early stages of the purine biosynthetic pathway and use of this mechanistic information to design specific inactivators of these enzymes. These inhibitors may function as antitumor or antiviral agents and may be of use to study the regulation of the purine biosynthetic pathway. The first basic objectives involve FGAR amidotransferase: (a) to examine in detail the mechanism of conversion of formylglycineamide ribonucleotide (FGAR) to formylglycineamidine ribonucleotide (FGAM) in order to obtain evidence for or against chemically and kinetically competent phospho-E or phospho-FGAR intermediates, (b) to examine the substrate specificity of FGAR amidotransferase and to use this information in conjunction with the mechanistic information to design potent specific inhibitors, (c) to prepare antibodies to native enzyme for use in determination of this enzyme's concentration in crude cell extracts and to determine if this antibody is cross-reactive with a larger molecular weight peptide in crude cells which may possess enzymatic activity of other early enzymes in the purine biosynthetic pathway, (d) to utilize FGAR amidotransferase and the antibody prepared to it as potential affinity columns in the isolation of phosphoribosylamine (PRA) amidotransferase. The second basic objective involves isolation and purification to homogeneity of aminoimidazole ribonucleotide (AIR) synthetase: a) to characterize this enzyme's molecular weight, pH optimum, kinetic properties and substrate specificty, b) to study the mechanism of this reaction by methods analogous to those described for the mechanistically similar FGAR amidotransferase, c) to use this mechanistic information to design a suicide inhibitor of this enzyme, d) to prepare antibodies to this enzyme to determine this enzyme's concentration in crude cell extract as well as to look for a larger polypeptide possessing other purine biosynthetic activities with which it might be cross-reactive, e) to use purified AIR synthetase and its antibody as potential affinity columns to isolate glycineamide ribonucleotide (GAR) synthetase. Our long range goals involve isolation of the first five enzymes in the purine biosynthetic pathway and eventual reconstitution of these enzymes in an attempt to find evidence, kinetic and biophysical, for a functional association.
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