Reversible Conjugation of Ethacrynic Acid with Glutathione and Human Glutathione S-Transferase PI-1

The reversibility of the conjugation reaction of the diuretic drug ethacrynic acid (EA), an a,~-unsaturated ketone, with glutathione and glutathione S-transferase PI-1 (GST PI-I) has been studied. When the glutathione conjugate of EA was incubated with a 5-fold molar excess of Nacetyi-L-cysteine or GST PI-I, a time-dependent transfer of EA to N-acetyI-L-cysteine or GST PI-1 was observed. With increasing pH, the pseudo first order rate constants of transfer of EA to N-acetyl-L-cysteine increased from 0.010 h -~ (pH 6.4) to 0.040 h -I (pH 7.4) and 0.076 h -~ (pH 8.4). From the fact that preincubation of GST PI-I with l-chioro-2,4-dinitrobenzene reduced the incorporation of p4C]EA from 0.94 _+ 0.21 (SD) to 0.16 + 0.02 mol EA/moi subunit and from automated Edman degradation of the major radioactive peptide isolated after pepsin digestion of the [~4C]EA-iabeled enzyme, it was concluded that the reaction of EA takes place with cysteine 47 of GST PI-1. When GST PI-1 was inactivated with a 5-fold molar excess of EA, adding an excess of glutathione resulted in full restoration of the catalytic activity in about 120 h. These findings may have several implications. Under normal physiological conditions the inhibition of GST PI-1 by covalent binding of EA would be reversed by glutathione, leaving reversible 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 be predominant, resulting in a completely different time course for the inhibition.