Structure and Mechanism of Yeast Orotate PRTase

Reed College, Portland OR

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Evidence to date clearly points to a Theorell-Chance mechanism for yeast orotate phosphoribosyltransferase (OPRTase), the enzyme responsible for the de novo production of all pyrimidine nucleotides in all organisms. Kinetic studies are proposed to put this hypothesis to a rigorous test. The principal investigator has perfected an overexpression system that allows large quantities of the recombinant native enzyme to be obtained. He has obtained crystals that diffract to 6A and will continue experiments aimed at producing diffraction-quality crystals of this enzyme complexed with appropriate ligands for OPRTase. Additionally, he will attempt to co-crystallize the enzyme with competent substrate analogs of PRPP, already synthesized by the principal investigator, and with analogs, which mimic the implicated oxocarbocation nature of the transition-state. Such analogs may, of course, be expected to act as biomedically active agents. Success in the crystallization experiments will allow, in collaborative studies, (an) X-ray structure(s) of OPRTase detailing the binding motif(s) of the various substrate(s) along with a structural map of the transition-state of the reaction catalyzed by this important enzyme. Such an analysis will go hand in hand with an understanding of the kinetic mechanism. The transition state analogs would also be evaluated as inhibitors of various PRTases. He also proposes the kinetic evaluation of the several mutant (single or double amino acid substitution(s) at or near the active site utilizing site directed mutagenesis) versions of yeast OPRTase designed to have reduced capacity to stabilize an oxocarbocation transition-state. It is expected that this research will shed new light on the mechanisms of action of a crucial enzyme. In general, mechanistic/structural studies such as these, often offer a powerful means to the rational design of antimetabolites - perhaps anticancer agents.