Inhibition and recognition studies on the glutathione-binding site of equine liver glutathione S-transferase.

Equine liver glutathione S-transferase has been shown to consist of two identical subunits of apparent Mr 25,500 and a pl of 8.9. Kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene as a substrate suggests a random rapid-equilibrium mechanism, which is supported by inhibition studies using glutathione analogues. S-(p-Bromobenzyl)glutathione and the corresponding N alpha-, CGlu- and CGly-substituted derivatives have been found, at pH 6.5, to be linear competitive inhibitors, with respect to GSH, of glutathione transferase. N-Acetylation of S-(p-bromobenzyl)glutathione decreases binding by 100-fold, whereas N-benzoylation and N-benzyloxycarbonylation abolish binding of the derivative to the enzyme. The latter effect has been attributed to a steric constraint in this region of the enzyme. Amidation of the glycine carboxy group of S-(p-bromobenzyl)glutathione decreases binding by 13-fold, whereas methylation decreases binding by 70-fold, indicating a steric constraint and a possible electrostatic interaction in this region of the enzyme. Amidation of both carboxy groups decreases binding significantly by 802-fold, which agrees with electrostatic interaction of the glutamic acid carboxy group with a group located on the enzyme.