Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25°C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min−1 and a of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 µM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11% for CDNB, 20% for ethacrynic acid, 2.5% for trans-stilbene oxide, and 2% for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.
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