The long term goal of this proposal is to add to the level of understanding of Glutathione S-transferases (GSTs). These enzymes play a central role in cellular resistance to anti-cancer drugs and in the metabolism and elimination of many other therapeutic agents. Development of inhibitors will be based on rational design, through detailed understanding of the chemical mechanisms of these enzymes. Studies will be done with the rat alpha 1-1 GST or the human alpha 1-1 isozyme, because these isozymes allow for direct spectroscopic studies of the active site tyrosine. The active site features that contribute to the unusually low pKa of the catalytic tyrosine of GSTs will be determined by site-directed mutagenesis and fluorescence and absorbance spectroscopies. The pKa of this residue plays a critical role in conjugation of glutathione to electrophilic substrates. It will be determined whether substrates and other ligands affect this pKa, and if so, a correlation between the pKa of this tyrosine in the presence of these ligands and kcat for the corresponding substrates will be sought. Similarly, the extent of solvation of this tyrosine has been proposed to be a critical determinant of GST catalysis. It will be determined whether this residue undergoes changes in solvent exposure in the presence of various ligands, and whether the extent of solvation correlates with kcat for the corresponding substrates. The subunit-subunit association of these dimeric enzymes will be studied with high-pressure techniques. The K-diss and delta-V for dissociation of the dimer will be determined for the wild type enzyme. Site - directed mutation of amino acids at the subunit interface will allow for determination of the specific contribution of individual residues to these thermodynamic parameters. With an understanding of the forces which control dimerization and the mechanisms of subunit recognition, rational design of agents that disrupt the assembly of functional GSTs will be possible.
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