Study on the Substrate Specificity of Arginine 13 Mutants in Human Glutathione S-Transferase

Abstract

In order to study he role of residue in the active site of glutathione S-transferase(GST), Arg13 residue in human GST P 1-1 was replaced with alanine, leucine or lysine by site-directed mutagenesis to obtain mutants R13A, R13L and R13K. These mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH towards electrophilic substrates. Our results suggest that Arg13 in human GST P1-1 contributes to the binding of electrophilic substrate, but it is not needed for the glutathione peroxidase activity and the steroid isomerase activity of human glutathione S-transferase P1-1.