This collaborative project will provide, for the first time, a detailed view of the structure and function of the phosphoribosyltransferases (PRTases), the enzymes involved in nucleotide formation. These enzymes are targets for anticancer chemotherapies, and their dysfunction is the origin of several metabolic disorders including Lesch-Nyhan disease. Despite their key metabolic role and pharmacological significance, no three- dimensional structures are known for these enzymes, and mechanistic analysis is limited to scattered observations on individual enzymes of the group. We have chosen orotate phosphoribosyltransferase (OPRTase) as the subject for our comprehensive study because of its small size and kinetic tractability. The enzyme catalyzes the nucleotide-forming step in de novo synthesis of pyrimidine nucleotides (UTP, TTP and CTP), and is a target for chemotherapy based on lethal synthesis from the anticancer drug 5- fluorouracil. Lack of OPRTase, either hereditary or drug-induced, leads to orotic acidurea. We have cloned, sequenced, and overexpressed the OPRTase (pyrE) gene from Salmonella typhimurium, and purified the resultant enzyme to homogeneity. The enzyme crystallizes readily in a form eminently suitable for x-ray crystallographic studies, and diffracts to better than 2.0 A resolution. A heavy atom derivative has been found. The system is poised for detailed investigations of the type described below. Our studies employ kinetic isotope effects and positional isotope exchange (PIX) to study the nature of the transition state for the reaction, providing important information for future inhibitor design. The mechanism is further explored using pH studies to localize enzymic proton acceptor/donor groups involved in catalysis. The three-dimensional structure of OPRTase will be determined to high resolution, and used to model the structure of the closely similar OPRTase domain of the pharmacologically important human UMP synthase. Using the structure and proposed mechanism, a series of modification/mutagenesis experiments is planned to explore the origin of the substrate specificity exhibited by the PRTases, and OPRTase in particular. We expect that the structural information generated will allow predictions of other PRTase structures, and that the principles of catalysis that we uncover will hold for other PRTases as well.
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