? 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 provide a major route for the metabolism and detoxification of alkylating agents. The enzymes exist as dimers of identical or closely related subunits. The three-dimensional structures of canonical GSH transferases reveal that each subunit is divided into two domains (I and II) that interact in a head-to-tail fashion to form a dimer. Previous results of this project have provided insight into the basic folding pathways and dimer assembly. In spite of this, little is known about the influence of specific molecular interactions on subunit or dimer stability. In addition, there is no information on the basic energetics of substrate and ligand binding to the active sites. In very recent work a bacterial GSH transferase (FosA) that catalyzes the addition of GSH to the antibiotic fosfomycin has been characterized. The enzyme is very specific toward fosfomycin and confers resistance to the antibiotic. It is the only GSH transferase known to be a metalloenzyme. The metal ion (Mn 2+) is directly involved in catalysis by providing electrophilic assistance in the reaction. Although FosA has a completely different fold as compared to the canonical enzymes it is also a dimer. Interestingly, preliminary evidence suggests that the metal ions are bound between the two subunits in the dimer. Thus, FosA provides a unique opportunity to investigate the influence of the metal ion on 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 for mutant class mu GSH transferases and the FosA protein; (ii) the characterization of the protein-ligand molecular 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. ? ?
| 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 |
