Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase π

Site-directed substitution mutations were introduced into a cDNA expression vector (pUC1207π) that encoded a human glutathione S-transferase π isozyme to non-conservatively replace four residues (Tyr7, Argl3, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to <2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7 → Phe mutant still retained 27 % of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.