PROBING THE ACTIVE-SITE OF ALPHA-CLASS RAT-LIVER GLUTATHIONE S-TRANSFERASES USING AFFINITY LABELING BY MONOBROMOBIMANE

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with K-m values of 0.63 mu M and 4.9 mu M for the mu-class isozymes 3-3 and 4-4, respectively, and 26 mu M for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu- class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold ) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The r ate of inactivation of 1-1 isozyme by mBBr is not decreased but, rathe r, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta- estradiol-3,17-disulfate (500 mu M) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated fo r 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after ther molysin digestion indicates that mBBr modification is equally distribu ted between two cysteine residues at positions 17 and 111. Modificatio n at these two sites is reduced equally in the presence of the added p rotectant, 17 beta-estradiol-3, 17-disulfate, suggesting that Cys 17 a nd Cys III reside within or near the enzyme's steroid binding sites. I n contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The differ ent reaction kinetics and modification sites by mBBr suggest that dist inct binding site structures are responsible for the characteristic su bstrate specificities of glutathione S-transferase isozymes.