Affinity labeling of rat glutathione S-transferase isozyme 1-1 by 17beta -iodoacetoxy-estradiol-3-sulfate.
Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.
Vargo
M A
MA
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Colman
R F
RF
D000345:Affinity Labels; D000818:Animals; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D051381:Rats; D017207:Rats, Sprague-Dawley; D013463:Sulfuric Acid Esters / Q000378:metabolism*
C423314:17-iodoacetoxyestradiol-3-sulfate; D000345:Affinity Labels; D007527:Isoenzymes; D013463:Sulfuric Acid Esters; D004958:Estradiol; D005982:Glutathione Transferase