AFFINITY LABELING OF CYS(111) OF GLUTATHIONE-S-TRANSFERASE, ISOENZYME-1-1, BY S-(4-BROMO-2,3-DIOXOBUTYL)GLUTATHIONE

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 1-1 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25-degrees-C results in a time-dependent inactivation of the enzyme. k(obs) exhibits a nonlinear dependence on S-BDB-G from 50 to 1200-mu-M, with a k(max) of 0.111 min-1 and K(I) = 185-mu-M. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, gives almost complete protection against inactivation by S-BDB-G. About 1.2 mol of [H-3]S-BDB-G/mol of enzyme subunit is incorporated when the enzyme is 85% inactivated, whereas 0.33 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when the enzyme has lost only 17% of its original activity. Modified enzyme, prepared by incubating glutathione S-transferase with [H-3]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with sodium borohydride, reacted with N-ethylmaleimide, and digested with alpha-chymotrypsin. Analysis of the chymotryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Cys111 as the amino acid whose reaction with S-BDB-G correlates with enzyme inactivation. It is concluded that Cys111 lies within or near the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 1-1.