INHIBITION OF HUMAN PLACENTA GLUTATHIONE TRANSFERASE P1-1 BY THE ANTIBIOTIC CALVATIC ACID AND ITS DIAZOCYANIDE ANALOG - EVIDENCE FOR MULTIPLE CATALYTIC INTERMEDIATES

The inhibition mechanism of the dimeric human placenta glutathione tra nsferase (GST) P1-1 by calvatic acid and the reaction intermediates, i .e. the diazocyanide analogue of calvatic acid, has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different molar ratios of inhibitor/GST P1-1 indicate that 1 mol calvatic acid inacti vates 1 mol GST P1-1, containing two catalytically equivalent active s ites. However, 2 mol of the diazocyanide analogue of calvatic acid ina ctivate 1 mol GST P1-1. Two disulfide bridges/dimer, probably between Cys47 and Cys101, have been formed during the reaction of GST P1-1 wit h calvatic acid and its diazocyanide analogue. The apparent second-ord er rate constants for GST P1-1 inactivation by calvatic acid and its d iazocyanide analogue are 2.4 +/- 0.3 M-1 s(-1) and (8.5+/-0.7)x10(3) M -1 s(-1), respectively. The reaction of calvatic acid with free L-cyst eine can be described by a simple process with an apparent second-orde r rate constant of (5.0 +/- 0.4)x10(1) M-1 s(-1). In contrast, a trans ient species occurs during the reaction of the diazocyanide analogue o f calvatic acid with free L-cysteine. Kinetics may be described by sec ond-order process [the rate constant being (8.0+/-0.5)x10(3) M-1 s(-1) ] followed by a first-order decay [the rate constant corresponding to (1.2+/-0.1)x10(1) s(-1)]. Calvatic acid represents an enzyme inhibitor acting much slower than its reaction intermediates (i.e. its diazocya nide analogue).