MECHANISMS OF ENZYMIC DETOXICATION REACTIONS

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The research is directed at the elucidation of the role and mechanisms of action of glutathione S-transferase and uridine-5 feet-diphosphoglucuronosyltransferase in the chemical and physical disposition of toxic foreign compounds (xenobiotics). Electrophilic substrates (arene oxides, Beta-lactones and Alpha, Beta-unsaturated compounds) and nucleophilic substrates (vicinal dihydrodiols of aromatic hydrocarbons) of known regio- and stereochemistry will be used to map the topology of the relatively nonspecific lipophilic binding sites of glutathione S-transferase and UDP-glucuronosyltransferase, respectively. As a prelude to detailed steady-state kinetic studies of glutathione S-transferase, several glutathione analogs, including Gamma-L-Glu-L-SerGly, Gamma-L-Glu-L-AlaGly, retroglutathione, and two methylene homologs, Beta-L-CysGly and Gamma-L-Glu-L-Cys-Beta-Ala, will be synthesized in quantity for use as alternative substrates and dead-end inhibitors. Chemical studies of the mechanism of catalysis will include attempts to trap transition states or intermediates along the reaction coordinate, in the reaction of glutathione with epoxides and arene oxides. The ionization behavior of bound glutathione will be studied by the pH dependence of the steady-state kinetics and a direct determination of the pKa of enzyme bound glutathione will be attempted by 13C-NMR spectroscopy. The ability of glutathione S-transferase to transport lipophilic solutes between populations of liposomes will be studied in order to assess its possible role as an intracellular xenobiotic transport protein. The chemical and kinetic mechanism of UDP-glucuronosyltransferase will be probed by Alpha-secondary deuterium kinetic isotope effects using uridine-5 feet-diphosphoglucuronate deuterated at the anomeric carbon and by steady-state kinetic studies employing a non-reactive methylene analog of UDP-glucuronate. Kinetic studies will be used to probe the phospholipid dependence and divalent metal ion dependence of the enzyme. The studies are designed to gain insight into the molecular details of the success or failure of glutathione S-transferase and UDP-glucuronosyltransferase in the metabolism of xenobiotics.