• Bulletin of the Korean Chemical Society
• /
• v.24 no.8
• /
• pp.1188-1192
• /
• 2003

In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr 108 residue in human GST P1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Tyr108 significantly affected $K_m^{CDNB}$ and $K_m^{ETA}$, whereas scarcely affected $K_m^{GSH}$. The substitutions of Tyr108 also significantly affected $I_{50}$ of ETA, an electrophilic substrate-like compound. The effect of these substitutions on kinetic parameters and the response to inhibition suggests that tyrosine 108 in hGST P1-1 contributes to the binding of the electrophilic substrate and a major determinant in the binding of CDNB is the aromatic ring of Tyr108, not its hydroxyl group.

• Bulletin of the Korean Chemical Society
• /
• v.26 no.3
• /
• pp.433-439
• /
• 2005

To gain further insight on the relationship between structure and functions of glutathione S-transferase (GST), the three tyrosine 108 mutants, Y108A, Y108F, and Y108W, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity, but approximately 63% increase of steroid isomerase activity toward ${\Delta}^5$–[androstene 3,17-dione. On the other hand, the substitution of Tyr 108 with phenylalanine resulted in decreases of $k_{cat}\;and\;k_{cat}/K_m{^{EPNP}}$ by 2 orders of magnitude, suggesting that Tyr 108 residue of hGSTP1-1 are considered to be important for the catalysis and the binding of the epoxide substrates. The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP, cumene hydroperoxide and ${\Delta}^5$–ndrostene 3,17-dione, but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB. We conclude from these results that Tyr 108 in hGST P1-1 plays very different roles depending upon the nature of the electrophilic substrates.