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Two forms of glutathione S-aryltransferase were purified from rat liver. The only differences noted between the two forms were in the chromatographic and electrophoretic properties, which permitted the separation of the two species. The molecular weights of the enzyme and its subunits were estimated as about 50000 and 23000 respectively. The steady-state kinetics did no follow Michaelis-Menten kinetics when one substrate concentration was kept constant while the second substrate concentration was varied. Several S-substituted GSH derivatives were tested as inhibitors of the enzymic reaction. The enzyme was inactivated by thiol-group reagents.

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...

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.