Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4-(succinimidyl)benzophenone]

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffi nity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1, A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the conc entration of the reagent, characterized by an apparent dissociation constan t of the enzyme-reagent complex (K-R) of 99 +/- 2 mu M and k(max) of 0.082 +/- 0.005 min(-1). Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenz ylglutathione) but not by steroids (Delta(5)-androstene-3,17-dione and 17 b eta-estradiol-3,17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, an d this reaction site is distinct from the substrate and nonsubstrate steroi d binding sites of the enzyme. About 1 mol of reagent is incorporated into I mol of enzyme dimer when the enzyme is completely inactivated, Met208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits, In order to evaluate the role of subunit intera ctions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-m odified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subuni ts were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2, The spec ific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GS T 1-1 is the indirect result of the interaction between the two enzyme subu nits and that this subunit interaction is absent in the heterodimer GST 1-2 .