The glutathione S-transferases (GSTs, EC 2.5.1.18) are ubiquitous in nature. They are part of the defense mechanism against environmental chemicals and some endogenous compounds. Recently, some GSTs have been implicated in the acquired resistance phenotypes in cancer chemotherapy. Two GSTs of the Ha (alpha) family, GST 1-1 and GST 2-2 which differ by only 11 amino acids out of 221 residues per subunit, display different but overlapping substrate specificities and product selectivity. for example, GST 1-1 and GST 2-2 catalyze the biotransformation of prostaglandin H2 (PGH2) into PGE2 and PGF2alpha and into PGD2 and PGF2alpha, respectively. They also have different specific activities against cumene hydroperoxide and t-butyl hydroperoxide. To understand the structural basis of GST catalysis and the different product selectivities of GST 1-1 and GST 2-2, this project seeks to exchange these substitutions in groups by making chimeric GSTs and also individually by site-directed mutagenesis.
The specific aims are: 1) To identify domains of GST 1-1 and/or GST 2-2 which are important in peroxidase activities and prostaglandin H2 activities by construction of chimeric GST genes and by site-directed mutagenesis. 2) To investigate roles of conserved amino acids in GST structure/function by chemical modification and site-directed mutagenesis. Each chimeric or mutant GST will be purified to electrophoretic homogeneity. Molecular characterization includes, but is not limited to, substrate specificities, prostaglandin H2 reactions, thermostability, catalytic efficiency, CD/ORD spectroscopy, and pH dependence of GST activities and kinetic constants. These results will be interpreted in terms of x-ray structural analyses. New experiments will be designed according to the x-ray data. Our current results suggest that flexibility in the structure/function of GSTs is an important asset in our defense against environmental chemicals. This flexibility allows us to suggest that acquired resistance to cancer chemotherapeutic agents in human patients may arise from qualitative changes in GSTs just as likely as from quantitative changes in GST expression. This project is very important in risk assessment of potential toxicity of environmental xenobiotics in vivo, and in understanding the enzymatic basis of interindividual variation in susceptibility to xenobiotics (i.e. pharmacogenetics).
