S-(4-BROMO-2,3-DIOXOBUTYL)GLUTATHIONE - A NEW AFFINITY LABEL FOR THE 4-4-ISOENZYME OF RAT-LIVER GLUTATHIONE-S-TRANSFERASE

S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200-mu-M) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25-degrees-C results in a time-dependent inactivation of the enzyme. The k(obs) exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000-mu-M, with a k(max) of 0.078 min-1 and K1 = 66-mu-M. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 1.3 mol of [H-3]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.48 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. 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 NaBH4, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified: Lys82-His-Asn-Leu-X-Gly-Glu-Thr-Glu-Glu-Glu-Arg93, in which X is modified Cys86, and Leu109-Gln-Leu-Ala-Met-CmCys-Y-Ser-Pro-Asp-Phe-Glu-Arg121, in which Y is modified Tyr115. Only the Lys82-Arg93 peptide was modified in the presence of S-hexylglutathione when the enzyme retained full activity. Cys86 is therefore regarded as a nonessential labeled residue, while Tyr115 is considered to be in the region of the active site. These results suggest that S-BDB-G functions as an affinity label at or near the active site of glutathione S-transferase and that modification of one site per enzyme subunit causes inactivation. It is proposed that the new compound, S-(4-bromo-2,3-dioxobutyl)glutathione, may have general applicability as an affinity label of other enzymes with glutathione binding sites.