Glutathione S-transferases are extremely important enzymes involved in the metabolism and detoxication of endogenous and foreign compounds. The research proposed is directed at elucidation of the mechanisms of action of glutathione S-transferases in the chemical disposition of the toxic xenobiotics. Four areas of investigation are proposed which include: (i) examination of the stereospecificity of the enzyme; (ii) synthesis of peptide analogues of glutathione; (iii) examination of the chemical and kinetic mechanisms of catalysis and; (iv) structural studies of the enzyme. The stereochemical aspects of substrate recognition by the enzyme will be investigated using arene oxide, Beta-lactone and alpha,beta-unsaturated substrates. Conformationally hindered arene oxides will be used to more closely examine the transition state and stereospecificity of oxirane ring opening. Structural aspects of the recognition of glutathione by the enzyme will be probed by synthesis and use of glutathione analogues including retroglutathione, glutathione homologues and diastereomers. Specifically labeled C13-glutathiones will be synthesized for use as C13-NMR probes of glutathione binding and determination of the pKa of the sulfhydryl group of enzyme-bound glutathione. The kinetic and chemical mechanisms of the enzyme will be studied using alternative substrate kinetics, pulse-chase techniques and trapping of potential intermediates in catalysis. Structural aspects of the enzyme and its active site will proceed through chemically labeling studies and attempts to grow X-ray quality crystals of the enzyme. The studies should lead to a better and possibly predictive understanding of the enzymes' involvement in the metabolism and detoxication of xenobiotics.
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