STRUCTURAL STUDIES ON HUMAN GLUTATHIONE-S-TRANSFERASE-PI - SUBSTITUTION MUTATIONS TO DETERMINE AMINO-ACIDS NECESSARY FOR BINDING GLUTATHIONE

In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi-enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120-pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST-pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, 168Y, L72F) showed a >95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a >20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.