Affinity Labeling of Pig Lung GlutathioneS-Transferase Pi by 4-(Fluorosulfonyl)benzoic Acid

The compound 4-(fluorosulfonyl)benzoic acid (4-FSB) functions as an affinity label of the dimeric pig lung pi class glutathioneS-transferase yielding a completely inactive enzyme. Protection against inactivation is provided by glutathione-based ligands, suggesting that the reaction target is near or part of the glutathione binding site. Radioactive 4-FSB is incorporated to the extent of 1 mol per mole of enzyme subunit. Peptide mapping revealed that 4-FSB reacts with two tyrosine residues in the ratio 69% Tyr7and 31% Tyr106. The ratio is not changed by the addition of ligands. The results suggest that only one of the tyrosine residues can be labeled in the active site of a given subunit; i.e., reactions with Tyr7and Tyr106are mutually exclusive. We propose that the difference in labeling of these tyrosine residues is related to their pKavalues, with Tyr7exhibiting the lower pKa. The modified enzyme no longer binds to aS-hexylglutathione–agarose affinity column, even when only one of the active sites contains 4-FSB; these results may reflect interaction between the subunits. We conclude that Tyr7and Tyr106of the pig lung class pi glutathioneS-transferase are important for function and are located at or close to the substrate binding site of the enzyme.