EXCEEDTHE SPACE PROVIDED. The glutathione_GSH) transferases catalyze the addition of GSH to-molecules bearing electrophilic functional groups. The canonical GSH transferases commonly found in mammals exhibit broad substrate specificity and 9rovide a major route for the metabolism and detoxification of alkylating agents. The enzymes exist as dimers o] identical or closely related subunits. The three-dimensional structures of canonical GSH transferases reveal thai _ach subunit is divided into two domains (I and II) that interact in a head-to-tail fashion to form a dimer. Previous :esults of this project have provided insight into the basic folding pathways and dimer assembly. In spite of thL, Little is known about the influence of specific molecular interactions on subunit or dimer stability. In addition, :here is no information on the basic energetics of substrate and ligand binding to the active sites. In very recent _,ork a bacterial GSH transferase (FosA) that catalyzes the addition of GSH to the antibiotic fosfomycin has been :haracterized. The enzyme is very specific toward fosfomycin and confers resistance to the antibiotic. It is the only 3SH transferase known to be a metalloenzyme. The metal ion (Mn 2 ) is directly involved in catalysis by providing zlectrophilic assistance in the reaction. Although FosA has a completely different fold as compared to the canonical _nzymes it is also a dimer. Interestingly, preliminary evidence suggests that the metal ions are bound between the :wo subunits in the dimer. Thus, FosA provides a unique opportunity to investigate the influence of the metal ion 3n protein folding and dimer assembly. The objectives of this application are to define the thermodynamic stabilities and elucidate specific molecular interactions associated with dimer stability of mutant canonical GSH transferases and the fosfomycin resistance protein, FosA, and to establish the energetics of substrate and ligand binding to these enzymes. The objectives of the research plan will be realized through completion of the following specific aims: (i) the elucidation of the thermodynamic stability of FosA and the folding pathways fol mutant class mu GSH transferases and the FosA protein; (ii) the characterization of the protein-ligand moleculm recognition processes of class mu GSH transferase and FosA by means of a thermodynamic analysis and; (iii) the development of a calorimetric enzyme assay for FosA for determining its reaction energetics. The research will be carried out by Prof. Heini Dirr and his research group in the Protein Structure-Function Research Programme al the Department of Biochemistry, University of Witwatersrand as an extension of NIH grant # R01GM30910-18. PERFORMANCE SITE ========================================Section End===========================================
| Kinsley, Nichole; Sayed, Yasien; Mosebi, Salerwe et al. (2008) Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1. Biophys Chem 137:100-4 |
| Thompson, Lawrence C; Walters, John; Burke, Jonathan et al. (2006) Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer. Biochemistry 45:2267-73 |
