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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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University of Wisconsin Madison
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Morar, Mariya; Hoskins, Aaron A; Stubbe, JoAnne et al. (2008) Formylglycinamide ribonucleotide amidotransferase from Thermotoga maritima: structural insights into complex formation. Biochemistry 47:7816-30
Hoskins, Aaron A; Morar, Mariya; Kappock, T Joseph et al. (2007) N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis. Biochemistry 46:2842-55
Anand, Ruchi; Hoskins, Aaron A; Stubbe, JoAnne et al. (2004) Domain organization of Salmonella typhimurium formylglycinamide ribonucleotide amidotransferase revealed by X-ray crystallography. Biochemistry 43:10328-42
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Hoskins, Aaron A; Anand, Ruchi; Ealick, Steven E et al. (2004) The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation. Biochemistry 43:10314-27
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Mathews, I I; Kappock, T J; Stubbe, J et al. (1999) Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway. Structure 7:1395-406
Thoden, J B; Kappock, T J; Stubbe, J et al. (1999) Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily. Biochemistry 38:15480-92
Li, C; Kappock, T J; Stubbe, J et al. (1999) X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution. Structure 7:1155-66

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