DESIGN OF MECHANISM BASED INHIBITORS OF DNA SYNTHESIS

这一建议是关于两种酶的机制的研究

This proposal is concerned with a study of the mechanism of two enzymes involved in DNA synthesis and use of this mechanistic information to design specific inactivators of these enzymes. The enzymes are ribonucleotide reductase (RDPR) and formylglycinamidine ribonucleotide (FGAM) synthetase. 1. Ribonucleotide reductases are uniquely responsible for the reduction of nucleotides to deoxynucleotides and have been divided into classes defined by the nature of their cofactor. This proposal is concerned with the class of reductases represented by the E. coli enzyme which requires 2 Fe+3 and an organic tyrosyl radical for activity. We have proposed a new mechanism for this reduction involving a radical cation intermediate. The basic objectives are: (1) to obtain evidence for, or against, a homolytic mechanism using rapid quench EPR spectroscopy and rapid scan spectroscopy; (2) to study the nature of the active site using the unique classes of suicide inhibitors; (3)to design several new classes of RDPR suicide inhibitors based on mechanistic information. 2. FGAM Synthetase is an enzyme involved in the early stages of purine biosynthesis. The basic objectives are (1) to examine in detail the mechanism of conversion of formyglycineamide ribonucleotide (FGAR) to FGAM in order to obtain evidence for, or against, chemically and kinetically competent phospho-E or phospho-FGAR intermediates; (2) To examine the substrate specificity of FGAM synthetase and to use this information in conjunction with the mechanistic information to design potent specific inhibitors; (3) 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; (4) to utilize FGAR amidotransferase and the antibody prepared to it as potential affinity columns in the isolation of phosphoribosylamine (PRA) amidotransferase. 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.