Inhibition of human placenta glutathione transferase P1-1 by the antibiotic calvatic acid and its diazocyanide analogue - Evidence for multiple catalytic intermediates

The inhibition mechanism of the dimeric human placenta glutathione transferase (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 inactivates 1 mol GST P1-1, containing two catalytically equivalent active sites. However, 2 mol of the diazocyanide analogue of calvatic acid inactivate 1 mol GST P1-1. Two disulfide bridges/dimer, probably between Cys47 and Cys101, have been formed during the reaction of GST P1-1 with calvatic acid and its diazocyanide analogue. The apparent second-order rate constants for GST P1-1 inactivation by calvatic acid and its diazocyanide 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-cysteine can be described by a simple process with an apparent second-order rate constant of (5.0 +/- 0.4)x10(1) M-1 s(-1). In contrast, a transient species occurs during the reaction of the diazocyanide analogue of calvatic acid with free L-cysteine. Kinetics may be described by second-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 diazocyanide analogue).