FACTORS AFFECTING THE INACTIVATION OF HUMAN PLACENTAL GLUTATHIONE S-TRANSFERASE-PI - THE KINETIC MECHANISM AND PH-DEPENDENCE OF SOLVATIONAL AND 1-CHLORO-2,4-DINITROBENZENE-MEDIATED INACTIVATION OF THE ENZYME

The kinetics of the inactivation of human placental GSH S-transferase π has been studied at 25° in the pH range 6.5 ≤ pH ≤ 9. At pH values ≤7.0 the inactivation of GSH S-transferase π incubated in the absence of GSH and (i) in the absence or (ii) in the presence of CDNB (0-1.5 × 10-3mol/dm3) exhibited pseudo first-order kinetics with kobs for (i) and (ii) approximately equal (∼0.002 sec-1). The extent of inactivation in (i) approached a limiting value of 50% at infinite dilution of the enzyme; while in the presence of CDNB the extent of inactivation approached 100%. At any given pH such that 7 < pH ≤ 9the pseudo first-order inactivation rate constant, kobs, exhibits a linear dependence on [CDNB] (Eqn 1): kobs = k1 + k2[CDNB] where k1 is invariant with pH and approximately equal to 0.002 sec-1. The first-(k1) and second-(k2)-order components of kobs suggest at least two mechanisms for the inactivation of GST by CDNB, these are: (i) a pH-invariant facilitation of solvational inactivation and (ii) a pH-dependent nucleophilic reaction of a thiol group (pka = 8.85 ± 0.08) at or spatially close to the active site of the enzyme. A mechanistic rationale for the enzyme functioning as a dimer is discussed in detail. © 1990.