ACTIVE-SITE TYROSYL RESIDUES ARE TARGETS IN THE IRREVERSIBLE INHIBITION OF A CLASS MU-GLUTATHIONE TRANSFERASE BY -(S-GLUTATHIONYL)-3,5,6-TRICHLORO-1,4-BENZOQUINONE

The mode of inactivation of glutathione S-transferase isoenzyme 3-3 fr om rat by the active site-directed inhibitor -(S-glutathionyl)-3,5,6-t richloro-1,4-benzoquinone (GSTCBQ) has been investigated by a combinat ion of site specific mutagenesis and mass spectrometric analysis of th e sites of reaction of the reagent with the enzyme. This very reactive reagent is shown to target 3 residues in or near the active site, inc luding the hydroxyl groups of Tyr-6 and Tyr-115 and the sulfhydryl gro up of Cys-114. Although the covalent attachment of one 2-(S-glutathion yl)dichloro-1,4-benzoquinonyl group/active site is sufficient to inact ivate the enzyme (<5% residual activity), the 1 mol of reagent appears to be distributed among all three target sites. Mutant enzymes in whi ch the reactive functional groups of these 3 residues have been indivi dually removed remain susceptible to GSTCBQ. Evidence from amino acid sequencing and peptide maps visualized by matrix-assisted laser desorp tion/ionization mass spectrometry suggests that both Tyr-6 and Tyr-115 are primary targets of the reagent in the native enzyme. Docking of a model of GSTCBQ in a model of the active site derived from the crysta l structure of the enzyme indicates that the trichlorobenzoquinonyl gr oup can be positioned so that both tyrosine hydroxyl groups can act as nucleophiles to add to the reagent or alternatively act as electrophi les to assist in the nucleophilic addition of the other. The reaction of GSTCBQ with Cys-114 appears to require a conformation different fro m that in the crystal structure.