Active-site tyrosyl residues are targets in the irreversible inhibition of a class Mu glutathione transferase by 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone.

The mode of inactivation of glutathione S-transferase isoenzyme 3-3 from rat by the active site-directed inhibitor 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GSTCBQ) has been investigated by a combination of site-specific mutagenesis and mass spectrometric analysis of the 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, including the hydroxyl groups of Tyr-6 and Tyr-115 and the sulfhydryl group of Cys-114. Although the covalent attachment of one 2-(S-glutathionyl)dichloro-1,4-benzoquinonyl group/active site is sufficient to inactivate the enzyme ( < 5% residual activity), the 1 mol of reagent appears to be distributed among all three target sites. Mutant enzymes in which the reactive functional groups of these 3 residues have been individually removed remain susceptible to GSTCBQ. Evidence from amino acid sequencing and peptide maps visualized by matrix-assisted laser desorption/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 crystal structure of the enzyme indicates that the trichlorobenzoquinonyl group can be positioned so that both tyrosine hydroxyl groups can act as nucleophiles to add to the reagent or alternatively act as electrophiles to assist in the nucleophilic addition of the other. The reaction of GSTCBQ with Cys-114 appears to require a conformation different from that in the crystal structure.