Probing the active site of α-class rat liver glutathione S-transferases using affinity labeling by monobromobimane

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Abstract
Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM 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 rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 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 thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In 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 different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.
Funding Information
  • United States Public Health Service (CA 66561)
  • National Research Service Award postdoctoral fellowship (F32 CA 66276)
  • United States Public Health Service.

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