Structural and functional consequences of haloenol lactone inactivation of murine and human glutathione S-transferase.

Mass spectrometric analysis of proteolysis products of haloenol lactone-modified glutathione S-transferase isozyme mGSTP1 indicates that the haloenol lactone 3-cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone is covalently attached to the protein at Cys-47. Comparisons of the extent of adduct formation with losses in enzymatic activity indicate that mGSTP1 exhibits greatest reactivity toward the haloenol lactone, followed by mGSTM1 and mGSTA3. Activities of mGSTP1 and mGSTM1 decrease in inverse proportion to haloenol lactone concentration, whereas modification had no apparent effect on catalytic activity of mGSTA3. Decreases in activity agree with the extent of protein modification observed in ESI mass spectra for mGSTP1 and mGSTM1 but not for mGSTA3. Kinetic studies employing recombinant human proteins with replacement of cysteine by serine at Cys-47 and Cys-101 indicate that rapid inactivation (t1/2 = 2 min) occurs only when residue 47 is cysteine. Mass spectra of C47S-hGSTP1 incubated with haloenol lactone demonstrate covalent attachment of a haloenol lactone-glutathione conjugate and suggest that an ester forms between the lactone and Ser-47. Therefore, we propose that initial opening of the lactone ring is promoted by Cys-47 through thioester formation between the lactone carbonyl and the Cys-47 sulfhydryl. Enol-keto tautomerization and enzyme-mediated hydrolytic cleavage of the thioester produces a reactive alpha-bromoketone which reacts a second time with Cys-47 and inactivates the enzyme. These results suggest that Pi class GSTs have thioesterase activity and that haloenol lactone inactivation occurs through an enzyme-mediated process.