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°C. Experiments performed at different molar ratios of inhibitor/GST P1-1 indicate that 1 mol calvatic acid inactivates 1 mol GST PI-1, containing two catalytically equivalent active sites. However, 2 rnol of the diazocyanide analogue of calvatic acid inactivate 1 mol GST PI-1. Two disulfide bridgesldimer, probably between Cys47 and CyslOl, 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 ± 10.3 M−1 s−1 and (8.5±0.7)×103M−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)×101 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 a second-order process [the rate constant being (8.0±0.5)×103 M−1 s−1 followed by a first-order decay [the rate constant corresponding to (1.2±0.1)×10−1 s−1]. Calvatic acid represents an enzyme inhibitor acting much slower than its reaction intermediates (i.e. its diazocyanide analogue).