MULTIFUNCTIONAL ROLE OF TYR-108 IN THE CATALYTIC MECHANISM OF HUMAN GLUTATHIONE TRANSFERASE P1-1 - CRYSTALLOGRAPHIC AND KINETIC-STUDIES ON THE Y108F MUTANT ENZYME

The possible role of the hydroxyl group of Tyr 108 in the catalytic me chanism of human glutathione transferase P1-1 has been investigated by means of site-directed mutagenesis, steady-state kinetic analysis, an d crystallographic studies. Three representative cosubstrates have bee n used, i.e. ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and 1-chloro-2,4-dinitrobenzene. In the presence of ethacrynic acid, t he enzyme follows a rapid equilibrium random bi-bi mechanism with a ra te-limiting step which occurs after the addition of the substrates and before the release of products. The replacement of Tyr 108 with Phe y ields a 14-fold decrease of k(cat), while it does not change appreciab ly the affinity of the H site for the substrate. In this case, it woul d appear that the role of the hydroxyl function is to stabilize the tr ansition state for the chemical step, i.e. the Michael addition of GSH to the electrophilic substrate. Crystallographic data are compatible with this conclusion showing the hydroxyl group of Y108 in hydrogen bo nding distance of the ketone moiety of ethacrynic acid [Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., & Parker, M . W. (1997) Biochemistry, 36, 576-585]. Moreover, no structural differ ences are observed between the Y108F mutant and the wild type, suggest ing that the removal of the hydroxyl group is solely responsible for t he loss of activity. A different involvement of Tyr 108 appears in the catalyzed conjugation of 7-chloro-4-nitrobenz-2-oxa- 1,3-diazole with GSH in which the rate-limiting step is of a physical nature, probably a structural transition of the ternary complex. The substitution of T yr 108 yields an approximately 7-fold increase of k(cat) and a constan t k(cat)/K-m(NBD-Cl) value. Lack of a critical hydrogen bond between 7 -chloro-4-nitrobenz-2-oxa- 1,3-diazole and Tyr 108 appears to be the b asis of the increased k(cat). In the 1-chloro-2,4-dinitrobenzene/GSH s ystem, no appreciable changes of kinetics parameters are found in the Y108F mutant. We conclude that Y108 has a multifunctional role in glut athione transferase P1-1 catalysis, depending on the nature of the ele ctrophilic cosubstrate.