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 pi has been studied at 25 degrees in the pH range 6.5 less than or equal to pH less than or equal to 9. At pH values less than or equal to 7.0 the inactivation of GSH S-transferase pi incubated in the absence of GSH and (i) in the absence or (ii) in the presence of CDNB (0-1.5 X 10(-3) mol/dm3) exhibited pseudo first-order kinetics with kobs for (i) and (ii) approximately equal (approximately 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 less than pH less than or equal to 9 the pseudo first-order inactivation rate constant, kobs, exhibits a linear dependence on [CDNB] (Eqn 1): kobs = k1 + k2 [CDNB] (1) 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.
Adams
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MRC Biomembrane Research Unit, University of Cape Town, Medical School, Observatory Republic of South Africa.
Sikakana
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D002478:Cells, Cultured; D004137:Dinitrochlorobenzene / Q000494:pharmacology*; D005260:Female; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010920:Placenta / Q000187:drug effects* / Q000201:enzymology
D004137:Dinitrochlorobenzene; D005982:Glutathione Transferase