Haloenol lactone is a new isozyme-selective and active site-directed inactivator of glutathione S-transferase

A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 microM) with murine Alpha, Mu, or Pi GST isozyme (1.0 microM) at pH 6.5, 37 degrees C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, approximately 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37 degrees C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 microM) was incubated with GST Pi isozyme (1.0 microM) in the presence of the competitive inhibitor S-hexylglutathione (10 microM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.