Abstract
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.
Highlights
They can exist as either homo- or heterodimers [1]
Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of ⌬5-androstene-3,17-dione and binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17-Iodoacetoxyestradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity
These results indicate that 17-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by ⌬5-androstene-3,17dione or glutathione
Summary
Materials—Frozen Harlan Sprague-Dawley rat livers were purchased from Pel Freez Biologicals, glutathione, S-hexylglutathione, Shexylglutathione-Sepharose, S-methylglutathione, Sephadex G-50, iodoacetic acid, ␣-chymotrypsin, 17-estradiol-3,17-disulfate, 17estradiol-3-sulfate, N,NЈ-dicyclohexylcarbodiimide, and 1-chloro-2,4dinitrobenzene were purchased from Sigma. 17-Estradiol-17-sulfate and ⌬5-androstene-3,17-dione were provided by Steraloids, Inc., and [1-14C]iodoacetic acid was purchased from Moravek Biochemicals. Reaction of 17-IES with Glutathione S-transferase, Isozyme 1-1— Glutathione S-transferase (0.2 mg/ml, 7.8 M enzyme subunits) was incubated in 0.1 M potassium phosphate buffer, pH 7.0, at 37 °C with various concentrations of 17-IES. Preparation and Separation of Proteolytic Digest of Modified Glutathione S-Transferase—Glutathione S-transferase (0.2 mg/ml) was incubated with 500 M 14C-labeled 17-IES at pH 7.0 under standard reaction conditions for 3 h, at which time the enzyme was maximally inactivated. The ester bond between the iodoacetic acid and estradiol-3-sulfate was subsequently hydrolyzed by adding 2 N NaOH to yield 0.2 N NaOH and incubating the enzyme digest at 25 °C for 2 h. The chymotryptic peptides were fractionated by a Varian 5000 LC equipped with a Vydac C18 reverse-phase column equilibrated with Solvent A (0.1% trifluoroacetic acid in water). Docking of 17-IES was done manually based on the energy minimized structure of 17estradiol-3,17-disulfate docked into isozyme 1-1 [15]
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