Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3 beta-(iodoacetoxy)dehydroisoandrosterone

3 beta-(Iodoacetoxy)dehydroisoandrosterone (3 beta-IDA), an analogue of the electrophilic substrate, Delta(5)-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3 beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3 beta-IDA concentration, yielding an apparent K-i of 21 mu M. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17 beta-estradiol-3,17-disulfate but, surprisingly, not by Delta(5)-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys(17) and Cys(111) are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys(111) on subunit A prevents modification of Cys(111) on subunit B and similarly for Cys(17). Molecular modeling analysis suggests that Cys(17) and Cys(111) are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme. (C) 1997 Academic Press.