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

Protein Science : a Publication of the Protein Society
L HuR F Colman

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 pre...Continue Reading

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Citations

Nov 6, 2001·Journal of Biochemical and Biophysical Methods·N Shirasu, Y Shimohigashi
Oct 24, 2003·Protein Science : a Publication of the Protein Society·Luis A Ralat, Roberta F Colman
Oct 30, 2010·Journal of Enzyme Inhibition and Medicinal Chemistry·Stanley MukanganyamaBerhanu Abegaz
May 5, 2006·Protein Science : a Publication of the Protein Society·Jennifer L Hearne, Roberta F Colman
Oct 1, 2005·Protein Science : a Publication of the Protein Society·Jennifer L Hearne, Roberta F Colman
Dec 19, 2001·Archives of Biochemistry and Biophysics·N E Pettigrew, R F Colman

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