Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism

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Abstract
Glutathione derivatives inhibit glutathione S-transferase A. The steady-state kinetics of this inhibition were investigated in detail by using S-ocytylglutathione, glutathione disulfide and S-(2-chloro-4-nitrophenyl)glutathione; the last compound is a product of the enzyme-catalyzed reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of 2 inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations was based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and increased with velocity. The established error structure was used as a basis for weighting in the regression and to construct confidence levels for the judgement of goodness-of-fit of rate equations fitted to experimental data. The results obtained support a steady-state random model for the mechanism of action of glutathione S-transferase A and exclude a number of simple kinetic models.