A Steady‐State‐Kinetic Random Mechanism for Glutathione S‐Transferase A from Rat Liver

Abstract

The steady-state kinetics of glutathione S-transferase A from rat liver were studied with the substrates glutathione and 3,4-dichloro-l-nitrobenzene varying from 1 pM to 5 mM and from 1 pM to 0.5 mM, respectively. The mathematical description of the experimental data required a rate law which was at least third degree with respect to concentration of each of the substrates. The rate equation of a generalized random sequential mechanism was fitted by a nonlinear regression program to the kinetic-data set and was found to be superior to other two-substrate models tested. Product inhibition studies with S-(2-chloro-4-nitrophenyl)-glutathione were also in accordance with a random sequential mechanism for the forward reaction. The second product of the reaction, chloride, was a very poor inhibitor. In the regression analysis of the data, the correlation of experimental error with velocity was taken into account by weighting. For this purpose an empirical error function was used.Binding of the product S-(2-chloro-4-nitrophenyl)-glutathione was studied by the method of equilibrium partition in an aqueous two-phase system. The binding could be described by a rectangular hyperbola which showed binding at a ratio of 1.95 molecules of product per molecule of enzyme at saturating product concentration. No cooperativity could be established by the binding studies and it is assumed that each of the two subunits of the enzyme can bind one molecule of S-(2-chloro-4-nitropheny1)-glutathione.The results of the different experiments were all in accordance with the proposition of a steadystate random sequential model for the mechanism of action of GSH S-transferase A, provided that kinetically significant enzyme-product complexes were introduced. In structural and functional terms the model has the following simple features: the two subunits of the enzyme are not assumed to display any cooperativity, and a subunit contains only one catalytically relevant binding site for GSH and GSH derivatives and one for the electrophilic substrate; these sites constitute subsites of the catalytically active center of the enzyme.Glutathione S-transferases constitute a group of ubiquitous enzymes [l] showing broad and overlapping substrate specificity [2]. Common to the substrates, many of which are mercapturic acid precursors, is that they all bear an electrophilic site and are conjugated to the obligatory second substrate GSH [3]. Some of the physical properties of highly purified glutathione S-transferases have been described (cf. [2,4,5]), but the establishment of their reaction mechanisms has remained unsettled. A comparative study of enzyme preparations from grass-grub and Abbreviation. C1,Ph-NO, , 3,4-dichloro-l -nitrobenzene.Enzyme. Glutathione S-transferase A or glutathione S-aryltransferase (EC 2.5.1.18).sheep liver has been made earlier by using 3,4-dichloro-1-nitrobenzene and GSH as substrates and under the assumption of a random sequential mechanism [6]. The kinetics of the enzymatic reaction between 1 -menaphthylsulphate and GSH hav...

The effect of ethanol on the steady‐state kinetics of glutathione S‐transferase a from rat liver

“…These results eliminate a trivial explanation of the non-Michaelian kinetics and lend further support to the steady-state kinetic random mechanism proposed for the enzyme [5,6].…”

“…Thus, the substrates do not prevent the binding of ethanol and the inhibition appears to be unspecific. More importantly, the present study shows unambiguously that the complex kinetic pattern [5,6] is not an effect of the presence of ethanol. This conclusion was reached by mathematical modelling of the authentic kinetic data obtained in the presence of ethanol as well as of the limit velocities obtained by extrapolation to zero ethanol concentration.…”

“…The enzyme was prepared and assayed as in [3,5]. The enzymatic reactions were started by addition of 3,4-dichloro-1-nitrobenzene dissolved in ethanol.…”

“…The enzymatic reactions were started by addition of 3,4-dichloro-1-nitrobenzene dissolved in ethanol. Nonlinear regression analysis of initial velocity data was as in [5]. Weighting factors were based on the analysis of the experimental error structure, which was estimated by replicate velocity determinations [8 3.…”

“…This enzyme is the only homogeneous GSH S-transferase which has been studied in detail by steady-state kinetic methods. The kinetics were found to be non-Michaelian [3,4] and the highly complex rate behaviour was interpreted by our group in terms of a steady-state random-order mechanism [5,6]. However, deviations from Michaelian kinetics may have many causes, and it is therefore essential to eliminate as many as possible of the alternative explanations.…”