The glutathione S-transferases are a group of detoxication enzymes that catalyze the nucleophilic addition of glutathione (GSH) to structurally diverse electrophilic substrates. This reaction is the single most important route for the metabolism and detoxication of alkylating agents in higher organisms. The catalytic versatility of these enzymes results from both the existence of a number of isoenzymes and the fact that each isoenzyme is endowed with a unique yet broad substrate specificity. The thesis of this proposal is that a thorough understanding of the participation of this family of proteins in the metabolism of drugs and xenobiotics must derive from a knowledge of the relationship between molecular structure and catalysis. The long term goal of the research proposed in this application is to develop an understanding of structure function relationships in catalysis by the GSH transferases and to explore methods for altering or designing new catalytic behavior. Mechanistic investigations designed to contrast the unique catalytic properties of isoenzymes 3-3 and 4-4 from rat liver will involve: (i) the elucidation of the role of sigma-complex intermediates in nucleophilic aromatic substitution reactions by rapid reaction techniques; (ii) spectroscopic characterization of the formation of a dead-end sigma-complex in single crystals of isoenzyme 3-3; (iii) the detection of active site functional group participation in Michael additions to alpha, beta-unsaturated ketones; (iv) a study of nucleophilic vinylic substitution reactions; and (v) a determination of the role of histidine and arginine residues in catalysis by 13C- and 15N-NMR spectroscopy of isotopically labeled enzymes and by site-directed mutagenesis. Altering the catalytic behavior of the GSH transferases to further define structure-function relationships will include: (i) the construction and expression of chimeric genes encoding hybrid isoenzymes with altered catalytic properties; (ii) Random mutagenesis of isoenzyme 3-3 followed by genetic selection of catalytic specificity toward new functional groups; and (iii) site directed mutagenesis of specific residues in the active site as revealed by X-ray crystallography. The three dimensional structure of isoenzyme 3-3 will be determined by X-ray diffraction techniques. Additional heavy atom derivatives will be prepared and used for phase determination and refinement. X-ray intensity data on substrate, product and intermediate complexes of the protein will be used to generate difference electron density maps for location and structural analysis of the active site region.
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