Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S‐transferase π

Abstract

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S‐transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S‐(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1‐chloro‐2,4‐dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8‐anilino‐1‐naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S‐Methylglutathione and S‐(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl‐bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys 45 and Cys 99 equally. Modification of Cys 45 is reduced in the presence of S‐methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys 99 is reduced in the presence of S‐(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys 99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S‐transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.