title: Affinity labeling of Cys111 of glutathione S-transferase, isoenzyme 1-1, by S-(4-bromo-2,3-dioxobutyl)glutathione.
abstract: 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 microM, with a kmax of 0.111 min-1 and KI = 185 microM. 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 [3H]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 [3H]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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
authors.forename: R M
authors.initials: RM
authors.lastname: Katusz
authors.forename: B
authors.initials: B
authors.lastname: Bono
authors.forename: R F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000345:Affinity Labels; D007527:Isoenzymes; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine