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

Abstract

Glutathione derivatives inhibit glutathione S-transferase A [cf. Biochem. J. (1975) 147, 513--522]. The steady-state kinetics of this inhibition have been investigated in detail by using S-octyglutathione, glutathione disulphide and S-(2-chloro-4-nitrophenyl)glutathione: the last compound is a product of the enzyme-catalused reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were found to be competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of two inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations has been based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and was found to increase 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.

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

“…However, there is a lack of agreement as to whether mammalian GSTs exhibit rapid-equilibrium or steady-state kinetics. In some cases, the random, rapid-equilibrium model provided the best fit to the data (Schramm et al, 1984;Ivanetich and Goold, 1989;Young and Briedis, 1989;Phillips and Mantle, 1991), whereas in others the kinetics were best described by the random, steady-state model (Jakobson et al, 1979;Ivanetich et al, 1990). It has been suggested Figure 10.…”

“…Bireactant models have been used to characterize the kinetic mechanisms of mammalian GSTs (Jakobson et al, 1979;Schramm et al, 1984;Ivanetich and Goold, 1989;Young and Briedis, 1989;Ivanetich et al, 1990;Phillips and Mantle, 1991). For mammalian GSTs, analyses of initialvelocity data with bireactant kinetic models have indicated that the kinetic mechanism is random and sequential.…”

“…2) because the presence of the squared terms in this model predicts nonlinear, reciprocal plots (Segel, 1975). Nonlinear, reciprocal initial-velocity plots have been observed for rat liver GST 2-2 and GST 3-3 (Jakobson et al, 1979;Ivanetich et al, 1990), and the initial-velocity data for these isozymes were fitted to the random, steady-state model. However, attempts to fit the initial-velocity data for GST B1/B2 to this model generated parameter values with high standard errors and multiple negative values.…”

Error structure as a function of substrate and inhibitor concentration in enzyme kinetic experiments

“…Fig. 5 in Jakobson et al, 1979). Proper units of the parameters can be deduced by considering that the standard error is expressed in /SM/min and the reactant concentrations in mm (cf.…”

“…The residuals obtained after fitting a suitable rate equation (see Fig. 5 in Jakobson et al, 1979) were divided into groups of six (as indicated by boxes in Fig. 5 of the present paper) and the local variance was calculated for each group of points.…”