THE INITIAL-RATE KINETICS OF MOUSE GLUTATHIONE-S-TRANSFERASE YFYF - EVIDENCE FOR AN ALLOSTERIC SITE FOR ETHACRYNIC-ACID

Mouse glutathione S-transferase GST YfYf (an orthologue of GST P or 7-7 in the rat and of GST pi in the human) was found to have a subunit M(r) of 24 500 and cross-reacted with anti(rat GST YfYf). N-Terminal analysis showed a close similarity to the rat, human and bovine orthologues. On isoelectric focusing the native enzyme had a pI of 8.3 and a pI of 7.3 in the presence of urea. Initial-rate studies with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates and inhibition studies with the product of the enzyme-catalyzed conjugation of CDNB and GSH, S-(2,4-dinitrophenyl)glutathione, indicated a rapid-equilibrium random mechanism for the enzyme. The diuretic drug ethacrynic acid was found to be simultaneously a competitive inhibitor and an uncompetitive activator of the enzyme (with CDNB as the substrate whose concentration was varied). By using a computer simulation program (EKPLOT) a model was developed that would explain the experimental data. It is proposed that ethacrynic acid can compete with CDNB at the active site but simultaneously bind to an allosteric site on the enzyme, causing an elevation in the V(max) for the conjugation of CDNB and GSH. The implications of such an activation mechanism for an enzyme potentially conjugating a range of xenobiotic compounds are discussed.