REVERSIBLE CONJUGATION OF ETHACRYNIC-ACID WITH GLUTATHIONE AND HUMAN GLUTATHIONE-S-TRANSFERASE P1-1

The reversibility of the conjugation reaction of the diuretic drug eth acrynic acid (EA), an alpha,beta-unsaturated ketone, with glutathione and glutathione S-transferase P1-1 (GST P1-1) has been studied. When t he glutathione conjugate of EA was incubated with a 5-fold molar exces s of N-acetyl-L-cysteine or GST P1-1, a time-dependent transfer of EA to N-acetyl-L-cysteine or GST P1-1 was observed. With increasing PH, t he pseudo first order rate constants of transfer of EA to N-acetyl-L-c ysteine increased from 0.010 h(-1) (pH 6.4) to 0.040 h(-1) (pH 7.4) an d 0.076 h(-1) (pH 8.4). From the fact that preincubation of GST P1-1 w ith 1-chloro-2,4-dinitrobenzene reduced the incorporation of [C-14]EA from 0.94 +/- 0.21 (SD) to 0.16 +/- 0.02 mol EA/mol subunit and from a utomated Edman degradation of the major radioactive peptide isolated a fter pepsin digestion of the [C-14]EA-labeled enzyme, it was concluded that the reaction of EA takes place with cysteine 47 of GST P1-1. Whe n GST P1-1 was inactivated with a 5-fold molar excess of EA, adding an excess of glutathione resulted in full restoration of the catalytic a ctivity in about 120 h. These findings may have several implications. Under normal physiological conditions the inhibition of GST P1-1 by co valent binding of EA would be reversed by glutathione, leaving reversi ble inhibition by the glutathione conjugate of EA and by EA itself as the main mechanism of inhibition; however, when glutathione levels are low the covalent inhibition might he predominant, resulting in a comp letely different time course for the inhibition.