The glutathione transferases catalyze the nucleophilic addition of the sulfur of glutathione (GSH) to a wide variety of endogenous and xenobiotic substrates bearing electrophilic functional groups. They are the single most important enzyme in the metabolism and detoxication of alkylating agents in mammals. In addition, they represent a superfamily of proteins with diverse and unexplored functions. It is the goal of this project to understand the role of glutathione transferases and their homologues in the biology of both prokaryotes and eukaryotes. This goal will be accomplished through the elucidation of the relationship between molecular structure and function. The investigations will focus on poorly understood aspects of the structure and catalytic mechanism of selected mammalian and bacterial enzymes.
Four specific aims will be pursued. First, the structure, dynamics and catalysis of glutathione transferases will be elucidated through; (i) the use amide-proton exchange mass spectrometry to probe the dynamics of specific structural elements in the class mu enzymes that control the rate of release of ligands (products) from the active site; (ii) extension of amide-proton exchange mass spectrometry into the millisecond time domain; and (iii) mapping of proton exchange kinetics to individual residues by tandem mass spectrometry. Second, selected GSI-I transferase homologues in the Escherichia coil proteome will be investigated to determine their unique biological roles. The functions of three members of the GSH transferase superfamily EGST, b2989 and yibF will be explored by; (i) cloning, expression and mechanistic characterization of the gene products; (ii) profiling the expression of the homologues in the E. coli proteome under different environmental conditions by two-dimensional difference gel electrophoresis and mass spectrometry; (iii) deletion of selected genes to determine phenotypic responses; and (iv) structure determination of the gene products. Third, class kappa and related GSI-I transferases will be investigated through (i) determination of the three dimensional structure of the class kappa enzyme from rat, (ii) analysis of the substrate specificity of the enzyme, and (iii) mechanistic analysis of the closely related 2-hydroxychromene-2-carboxylate isomerase in the naphthalene catabolic pathway of Pseudomonas putida. Finally, mechanistic and structural investigations of the microsomal enzyme will be extended by; (i) analysis of the unique mechanistic behavior of the activated enzyme; (ii) an exploration of the structure and dynamics of the native and activated forms of the enzyme in detergent solution by amide-proton exchange mass spectrometry; and (iii) amide-proton exchange analysis of two-dimensional crystals by mass spectrometry.
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