Affinity labeling of rat GST A1-1 by 3β-(iodoacetoxy)dehydroisoandrosterone

3β-(Iodoacetoxy)dehydroisoandrosterone (3β-IDA), a reactive steroid analogue, functions as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent inactivation is observed upon incubation of enzyme with 3β-IDA at pH 7.5 and 37°C. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Protection against inactivation is provided by the non-substrate steroid ligand 17β-estradiol-3,17-disulfate but not by Δ⁵-androstene 3,17-dione or any other electrophilic substrate tested. These results suggest that reaction occurs within a non-substrate steroid site, which is distinguishable from the electrophilic substrate binding site. Two cysteines, Cys¹⁷ and Cys¹¹¹, are modified in equal amounts, although the average reagent incorporation is 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer upon complete inactivation. Molecular modeling suggests that Cys¹⁷ and Cys¹¹¹ are located in the non-substrate steroid binding site, within the cleft between the two subunits of the dimeric enzyme.