A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase.
Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.
Ivanetich
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Biomolecular Resource Centre, University of California, San Francisco 94143-0541.
Goold
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D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002384:Catalysis; D003198:Computer Simulation; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D005982:Glutathione Transferase; D005978:Glutathione