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Affinity Labeling of Rat Glutathione S-Transferase Isozyme 1-1 by 17β-Iodoacetoxy-estradiol-3-sulfate

2001; Elsevier BV; Volume: 276; Issue: 3 Linguagem: Inglês

10.1074/jbc.m008212200

1083-351X

Melissa A. Vargo, Roberta F. Colman,

Estrogen and related hormone effects

Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Δ5-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17β-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathioneS-transferase to a limit of 60% residual activity. The rate constant for inactivation (k obs) exhibits a nonlinear dependence on reagent concentration withK I = 71 μm andk max = 0.0133 min−1. Complete protection against inactivation is provided by 17β-estradiol-3,17-disulfate, whereas Δ5-androstene-3,17-dione andS-methylglutathione have little effect onk obs. These results indicate that 17β-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Δ5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys17 is the only enzymic amino acid modified. Covalent modification of Cys17 by 17β-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 Arg14 of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label. Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Δ5-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17β-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathioneS-transferase to a limit of 60% residual activity. The rate constant for inactivation (k obs) exhibits a nonlinear dependence on reagent concentration withK I = 71 μm andk max = 0.0133 min−1. Complete protection against inactivation is provided by 17β-estradiol-3,17-disulfate, whereas Δ5-androstene-3,17-dione andS-methylglutathione have little effect onk obs. These results indicate that 17β-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Δ5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys17 is the only enzymic amino acid modified. Covalent modification of Cys17 by 17β-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 Arg14 of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label. glutathioneS-transferase 3β-(iodoacetoxy)dehydroisoandrosterone 17β-iodoacetoxy-estradiol-3-sulfate high pressure liquid chromatography Glutathione S-transferases (GST)1 (EC 2.5.1.18) constitute a family of detoxification enzymes that are involved in the metabolism of endogenous and xenobiotic compounds (1Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (969) Google Scholar, 2Mannervik B. Danielson U.H. CRC Crit. Rev. Biochem. 1988; 23: 283-337Crossref PubMed Scopus (1688) Google Scholar, 3Wilce M.C.J. Parker M.W. Biochim. Biophys. Acta. 1994; 1205: 1-18Crossref PubMed Scopus (548) Google Scholar, 4Pickett C.B. Lu A.Y.H. Annu. Rev. Biochem. 1989; 58: 743-764Crossref PubMed Scopus (553) Google Scholar). They catalyze the conjugation reaction of glutathione to a wide variety of electrophilic substrates. These conjugation products are more water-soluble than the xenobiotic substrates, and they can be further degraded or transported out of the cell. GlutathioneS-transferases have been found in elevated levels within cancerous tumors and have been implicated in the development of resistance to anti-cancer drugs (5Hayes J.D. Pulford D.J. CRC Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar). The cytosolic enzymes are now grouped into seven classes and within a particular class they can exist as either homo- or heterodimers (1Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (969) Google Scholar). There are crystal structures to represent most of the classes (6Ji X. Zhang P. Armstrong R.N. Gilliland G.L. Biochemistry. 1992; 36: 10169-10184Crossref Scopus (378) Google Scholar, 7Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dirr H.W. Huber R. Gilliland G.L. Armstrong R.N. Ji X. Board P.G. Olin B. Mannervik B. Jones T.A. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (415) Google Scholar, 8Reinemer P. Dirr H.W. Ladenstein R. Schaffer J. Gallay O. Huber R. EMBO J. 1991; 10: 1997-2005Crossref PubMed Scopus (332) Google Scholar, 9Ji X. Tordova M. O'Donnell R. Parsons J.F. Hayden J.B. Gilliland G.L. Zimniak P. Biochemistry. 1997; 36: 9690-9702Crossref PubMed Scopus (91) Google Scholar, 10Oakley A.J. Rossjohn J. Lo Bello M. Caccuri A.M. Federici G. Parker M.W. Biochemistry. 1997; 36: 576-585Crossref PubMed Scopus (121) Google Scholar, 11Wilce M.C.J. Board P.G. Feil S.C. Parker M.W. EMBO J. 1995; 14: 2133-2143Crossref PubMed Scopus (218) Google Scholar, 12Ji X. Von Rosenvinge E.C. Johnson W.W. Tomarev S.I. Piatigorsky J. Armstrong R.N. Gilliland G.L. Biochemistry. 1995; 34: 5317-5328Crossref PubMed Scopus (216) Google Scholar). Each subunit of the dimer contains a glutathione-binding site and a xenobiotic site that can accommodate a wide variety of compounds. Isozyme 1-1, 2Glutathione S-transferase, isozyme 1-1, is designated as the rGSTA1,2 isozyme in the nomenclature of Hayes and Pulford (5Hayes J.D. Pulford D.J. CRC Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar). a member of the α class, efficiently catalyzes the isomerization reaction of Δ5-androstene-3,17-dione to Δ4-androstene-3,17-dione, which it binds at the substrate steroid site (13Benson A.M. Talalay P. Keen J.H. Jakoby W.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 158-162Crossref PubMed Scopus (156) Google Scholar). In addition to this site, isozyme 1-1 also has a nonsubstrate steroid binding site that is located in the cleft between the two subunits (14Hu L. Colman R.F. Biochemistry. 1997; 36: 1635-1645Crossref PubMed Scopus (11) Google Scholar, 15Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar). This site has been proposed to fulfill a transport function (5Hayes J.D. Pulford D.J. CRC Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar) or to act in controlling levels of steroids in target organs (16Listowsky I. Structure and Function of Glutathione S-transferases. CRC Press, Boca Raton, FL1993: 199-209Google Scholar). The nonsubstrate site has a preference for steroid sulfates, which is illustrated by the more potent inhibitory effect of 17β-estradiol-3,17-disulfate as compared with that of 17β-estradiol. However, previous work in this laboratory (aimed at locating the nonsubstrate site) used the affinity label 3β-(iodoacetoxy)dehydroisoandrosterone (3β-IDA) (shown in Fig. 1), which is structurally related both to substrates of the enzyme, such as Δ5-androstene-3,17-dione, and to inhibitors of the enzyme, such as Δ5-androstene-3β,17β-diol disulfate and 17β-estradiol-3,17-disulfate. The 3β-IDA modified Cys17 and Cys111 equally with an incorporation of 1 mol of reagent/mol enzyme subunit; analysis of molecular models suggested that the binding site of 3β-IDA is located in the cleft between the subunits (15Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar). Based on the previous data, we have now designed a more specific affinity label for the nonsubstrate steroid site: 17β-iodoacetoxy-estradiol-3-sulfate (17β-IES). This new compound features the negatively charged sulfate that should enhance and direct its binding and a reactive iodoacetoxy group at a position at the opposite end of the molecule from that of 3β-IDA (Fig. 1). The iodide can be displaced from the iodoacetoxy group by nucleophilic attack by the side chains of several amino acids including Cys, Asp, Lys, Met, and His (17Wilchek M. Givol D. Methods Enzymol. 1977; 46: 153-157Crossref PubMed Scopus (36) Google Scholar). In this paper, we demonstrate that this affinity label reacts specifically with Cys17 at a single subunit of the enzyme dimer. Molecular modeling studies support the location of the nonsubstrate binding site within the cleft and the contribution of the sulfate moiety in orienting the ligand within the cleft. A preliminary version of this work has been presented (18Vargo M.A. Colman R.F. Biochemistry. 2000; 39: 1544Google Scholar). Frozen Harlan Sprague-Dawley rat livers were purchased from Pel Freez Biologicals, glutathione,S-hexylglutathione, S-hexylglutathione-Sepharose,S-methylglutathione, Sephadex G-50, iodoacetic acid, α-chymotrypsin, 17β-estradiol-3,17-disulfate, 17β-estradiol-3-sulfate,N,N′-dicyclohexylcarbodiimide, and 1-chloro-2,4-dinitrobenzene were purchased from Sigma. 17β-Estradiol-17-sulfate and Δ5-androstene-3,17-dione were provided by Steraloids, Inc., and [1-14C]iodoacetic acid was purchased from Moravek Biochemicals. Bio-Rad Laboratories provided Protein Assay Dye Reagent, and Liquiscint was purchased from National Diagnostics. Glutathione S-transferase isozyme 1-1 was purified from rat livers using affinity column chromatography on S-hexylglutathione-Sepharose (19Wang J. Barycki J.J. Colman R.F. Protein Sci. 1996; 5: 1032-1042Crossref PubMed Scopus (18) Google Scholar). Values of ε270 nm = 22,000 m−1cm−1 (20Katusz R.M. Colman R.F. Biochemistry. 1991; 30: 1230-1238Crossref Scopus (42) Google Scholar) and molecular weight of 25,500 per subunit (2Mannervik B. Danielson U.H. CRC Crit. Rev. Biochem. 1988; 23: 283-337Crossref PubMed Scopus (1688) Google Scholar) for GST 1-1 were used to calculate the enzyme concentration. 17β-IES was synthesized from 17β-estradiol-3-sulfate and iodoacetic acid by procedures based on the method of Pons et al. (21Pons M. Nicolas J.C. Boussioux A.M. Descombs B. Crastes de Paulet A. FEBS Lett. 1973; 36: 23-30Crossref PubMed Scopus (21) Google Scholar). One molar equivalent of 17β-estradiol-3-sulfate, 1.1 molar equivalents of iodoacetic acid, and 2 molar equivalents of dicyclohexylcarbodiimide were combined in 15 ml of cellosolve. (For the radioactively labeled compound before addition to the reaction mixture, 125 μCi of radioactive iodoacetic acid was added to 0.83 mmol of unlabeled iodoacetic acid in a total of 5 ml.) The reaction was initiated by the addition of a catalytic amount of pyridine (250 μl), and the reaction mixture was allowed to stir at room temperature for 1.5 h. The reaction was stopped by the addition of 3 ml of distilled water, and the mixture was centrifuged to remove the insoluble dicyclohexylurea. The organic layer, containing 17β-IES, was lyophilized. The product was resuspended in 100 μl of acetonitrile and was brought to a final volume of 1 ml by the addition of distilled water. The 17β-IES was purified by HPLC using a Varian 5000LC equipped with a Vydac C18 column (1 × 25 cm) and a UV-100 detector. The solvent system used was H2O (Solvent A) and acetonitrile (Solvent B). The column was equilibrated with solvent A containing 10% solvent B. After 10 min at 10% solvent B, a linear gradient was run to 100% B in 90 min at a flow rate of 1 ml/min. The effluent was monitored at 275 nm and 17β-IES eluted at ∼28 min. For comparison, the starting material, 17β-estradiol-3-sulfate, elutes at ∼23 min. For the radioactively labeled compound, the specific radioactivity was 2.17 × 1011 cpm/mol. The product has a UV absorption spectrum with a maximum at 260 nm and a shoulder at 270 nm. The extinction coefficient at 260 nm was measured to be 1810m−1 cm−1, with the concentration determined from the specific radioactivity. Enzymatic activity was measured by using a Hewlett Packard 8453 UV-VIS Spectrophotometer and monitoring the formation of the glutathione (2.5 mm in assay) and 1-chloro-2,4-dinitrobenzene (1 mm in assay) conjugate at 340 nm (Δε = 9.6 mm−1cm−1) in 0.1 m potassium phosphate buffer, pH 6.5, at 25 °C according to Habig et al. (22Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). Glutathione S-transferase (0.2 mg/ml, 7.8 μm enzyme subunits) was incubated in 0.1 mpotassium phosphate buffer, pH 7.0, at 37 °C with various concentrations of 17β-IES. Control enzyme samples were incubated under the same conditions but without 17β-IES. At various time points, an aliquot was removed from the incubation mixture, diluted, and assayed (30 μl) for residual activity. Glutathione S-transferase (0.2 mg/ml) was incubated with 500 μm [14C]17β-IES at pH 7.0 under standard reaction conditions. Aliquots were withdrawn at various times, and excess reagent was removed by the gel centrifugation method using two successive Sephadex G-50 columns (5 ml) equilibrated with 0.1 m potassium phosphate buffer, pH 7.5 (23Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar). The protein concentration in the filtrate was determined using the Bio-Rad protein assay, based on the Bradford method, using a Bio-Rad 2550 RIA plate reader with a 600-nm filter (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Unmodified GST 1-1 was used to generate the standard concentration curve. The amount of reagent present was determined by radioactivity using a Packard 1500 Liquid scintillation counter. Incorporation was expressed as mol 17β-IES/mol of enzyme subunit. Glutathione S-transferase (0.2 mg/ml) was incubated with 500 μm14C-labeled 17β-IES at pH 7.0 under standard reaction conditions for 3 h, at which time the enzyme was maximally inactivated. Excess reagent was removed as described above. Solid guanidine HCl was added to make a 5 m guanidine-HCl solution and was incubated for 1 h at 37 °C to denature the protein, followed by treatment with 10 mm N-ethylmaleimide at 25 °C for 30 min to block free cysteine residues. The solution was then dialyzed against 6 liters of 10 mm ammonium bicarbonate, pH 8, at 4 °C with one change for a total of 18 h, after which the sample was lyophilized. The enzyme was solubilized by adding 250 μl of 8 m urea in 10 mm ammonium bicarbonate, pH 8.0, and incubating at 37 °C for 1 h. The solution was then diluted with 10 mm ammonium bicarbonate to bring the final concentration of urea to 2 m. Chymotrypsin was added (10% w/w) at 2 h intervals while incubating at 37 °C. 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 then incubating the enzyme digest at 25 °C for 2 h. The solution was then neutralized by adding HCl to yield 0.2 n. The solution was filtered through a 0.45 μm filter, with no loss of radioactivity and was subjected to HPLC. 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). At a flow rate of 1 ml/min, the peptides were separated by a linear gradient from 0% to 20% Solvent B (0.1% trifluoroacetic acid in acetonitrile) in 100 min followed by a linear gradient to 100% Solvent B in 30 min. The eluate was monitored by A 220, and 1-ml fractions were collected. An aliquot (300 μl) from each fraction was added to 5 ml of Liquiscint to test for radioactivity. The amino acid sequences of purified peptides were determined on an Applied Biosystems model 470A gas phase protein/peptide sequencer, equipped with a model 120A phenylthiohydantoin analyzer. Molecular modeling was conducted using the Insight II modeling package from Molecular Simulations, Inc. on an Indigo 2 work station from Silicon Graphics. The model of rat GST 1-1 was constructed as described previously (19Wang J. Barycki J.J. Colman R.F. Protein Sci. 1996; 5: 1032-1042Crossref PubMed Scopus (18) Google Scholar) based on the known crystal structure of human liver isozyme 1-1 (1GUH). The structure of 17β-IES was constructed using the Builder module. Docking of 17β-IES was done manually based on the energy minimized structure of 17β-estradiol-3,17-disulfate docked into isozyme 1-1 (15Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar). Incubation of rat GST 1-1 (0.2 mg/ml, 7.8 μmenzyme subunits), with 300 μm 17β-IES, when assayed with 1-chloro-2,4 dinitrobenzene, results in a time-dependent loss of enzyme activity that reaches a limit of 60% of the original activity, as is illustrated in Fig.2 A. After 180 min, excess reagent was removed, and a second addition of 300 μm17β-IES was added; no further decrease in activity occurred. Because the activity levels off at 60% at long incubation times and over a range of 17β-IES concentrations, the data were calculated using 60% as the end point (Fig. 2 B). Control enzyme incubated under the same conditions but with no reagent present shows no loss of activity. The k obs for inactivation was calculated from the slope of ln([E t −E ∞]/[E 0 −E ∞]) versus time whereE t is the enzyme activity at time t,E 0 is the original enzyme activity, andE ∞ is the enzyme activity at long times, which is equal to 0.6 (E 0). The reaction obeys pseudo-first order kinetics with a rate constant of 0.0125 min−1 (Fig. 2 B). GST 1-1 (0.2 mg/ml, 7.8 μm enzyme subunits) was incubated with 20–300 μm of 17β-IES as described above, to determine the rate of inactivation at various reagent concentrations (Fig.3). The apparent rate constantk obs exhibits a nonlinear dependence on reagent concentration. This type of curve is typical of an affinity label, suggesting that a reversible enzyme-reagent complex is formed prior to the irreversible modifcation of the enzyme (26Colman, R. F. (1997) in Protein Function: A Practical Approach, 2nd Ed, Chapter 16, pp. 155–183, Oxford University Press, New YorkGoogle Scholar). The curve can be described by the equation k obs =k max/(1 + K I /[17β-IES]), where K I is the apparent dissociation constant of the enzyme-reagent complex, and k max is the maximum rate of inactivation at saturating concentrations of the reagent. A least squares fit of the observed data yieldsK I = 71.4 μm andk max = 0.0133 min−1. Various ligand analogues were added to the reaction mixture to determine whether they could protect against the inactivation of the enzyme by 100 μm 17β-IES. The results, given in Table I, are expressed as k +L/k −L, wherek +L is the rate constant for inactivation in the presence of a particular ligand, and k −L is the rate constant for inactivation in the absence of a particular ligand. Glutathione derivatives (Table I, lines 2 and 3) offer some protection, with the protective effect increasing with an increase in alkyl chain length. The 5 mm concentrations used are sufficient to saturate the glutathione site, yetk +L/k −L does not decrease below 0.37. These results suggest that the target site of 17β-IES is near the glutathione site but distinct from it. Electrophilic substrates, such as Δ5-androstene-3,17-dione (Table I, line 4), do not provide any protection. In contrast, including steroid sulfates, such as 17β-estradiol-3,17-disulfate, cause a striking decrease in the observed inactivation rate constant (lines 5–10). Because these steroid sulfates are known to bind at a nonsubstrate steroid site (15Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar), the results indicate that 17β-IES is reacting within this nonsubstrate steroid binding site.Table IEffects of enzyme ligands on the inactivation of glutathione S-transferase by 300 μm 17β-IESLigand addedk +L/k −L 1-ak+L/k −Lwas determined by the ratio of initial inactivation rate with ligand present to that observed in the absence of ligand. 1.None1.00 2.S-Methyl glutathione (5 mm)0.74 3.S-Hexyl glutathione (5 mm)0.37 4.Δ5-Androstene-3,17-dione (500 μm)1.00 5.17β-Estradiol-3-sulfate (100 μm)0.40 6.17β-Estradiol-3-sulfate (500 μm)0.25 7.17β-Estradiol-17-sulfate (100 μm)0.45 8.17β-Estradiol-17-sulfate (500 μm)0.14 9.17β-Estradiol-3,17-disulfate (100 μm)0.3010.17β-Estradiol-3,17-disulfate (500 μm)0.00The inactivation reaction was conducted at 37 °C in 0.1m potassium phosphate buffer, pH 6.5.1-a k+L/k −Lwas determined by the ratio of initial inactivation rate with ligand present to that observed in the absence of ligand. Open table in a new tab The inactivation reaction was conducted at 37 °C in 0.1m potassium phosphate buffer, pH 6.5. GST 1-1 (0.2 mg/ml) was incubated with 300 μm[14C]17β-IES. A time-dependent incorporation of [14C]17β-IES was observed concomitant with the decrease in enzyme activity. A plot of the percentage of maximum inactivation versus net incorporation (Fig.4) extrapolates to ∼0.5 mol of14C-labeled reagent incorporated per mol of enzyme subunit or 1 mol/enzyme dimer at 100% of maximum inactivation. Maximally inactivated GST 1-1 was prepared and digested with chymotrypsin. The digest was fractionated by HPLC using a reverse-phase column (C18) equilibrated with 0.1% trifluoroacetic acid and an acetonitrile gradient (Fig.5). One radioactive peptide peak was observed on HPLC. Because the ester linkage of 17β-IES (Fig. 1) was hydrolyzed before the digest was applied to HPLC, the steroid moiety was removed, and the peptide is expected to be labeled with the radioactive carboxymethyl group. The fractions corresponding to this peak were pooled, lyophilized, and subjected to gas phase amino acid sequencing. The results are shown in TableII. The sequence Glu-Xaa-Ile-Arg-Trp corresponds to residues 16–20 in the known amino acid sequence. None of the common phenylthiohydantoin derivatives was detected in cycle 2; instead, there was a peak with a retention time between that of phenylthiohydantoin-Ser and phenylthiohydantoin-Asn. This peak corresponds to that of a phenylthiohydantoin-carboxymethylcysteine standard, indicating that a Cys in this position had been modified. Thus, Cys17 of GST1-1 is the amino acid target of 17β-IES.Table IIRepresentative sequence of modified peptide (Peak I) isolated from the chymotryptic digest of 14 C-labeled glutathione S-transferase 1–1, as illustrated by Fig. 5CycleAmino acidpmol1Glu (141)2Xaa 2-aRetention time is 7.8 min between PTH-Asn and PTH-Ser.3Ile (37)4Arg (34)5Trp (14)2-a Retention time is 7.8 min between PTH-Asn and PTH-Ser. Open table in a new tab 17β-Iodoacetoxy-estradiol-3-sulfate acts as an affinity label of rat liver glutathione S-transferase isozyme 1-1. Upon incubation of the enzyme with 17β-IES, a time-dependent loss of activity is observed, yielding a maximum loss of 40% of the original activity. The rate of inactivation exhibits nonlinear dependence on reagent concentration, as is typical of an affinity label, for which an enzyme-reagent complex forms prior to irreversible modification. Partial protection against inactivation is provided by glutathione derivatives; long chain derivatives, such asS-hexylglutathione, provide more protection than do shorter chain derivatives, like S-methylglutathione, indicating that 17β-IES is binding in a site close to the glutathione site but not within the site. Electrophilic substrate analogues, such as Δ5-androstene-3,17-dione, do not offer any protection, demonstrating that 17β-IES does not bind within the electrophilic substrate site. Steroid sulfates are most effective in protecting against inactivation of GST, 1-1, with 17β-estradiol-3,17-disulfate providing complete protection. These results indicate that 17β-IES is binding and reacting within the nonsubstrate steroid binding site. Upon maximum inactivation, about 0.5 mol of reagent is incorporated per mol enzyme subunit or 1 mol of 17β-IES/enzyme dimer, and Cys17 is the only amino acid that is modified. In previous work, based on the crystal structure of glutathioneS-transferase from the parasitic worm Schistosoma japonica in complex with praziquantel, an anti-schistosomal drug bound in the cleft between the subunits, only 1 mol of praziquantel is bound per mol of enzyme dimer (27McTigue M.A. Williams D.R. Tainer J.A. J. Mol. Biol. 1995; 246: 21-27Crossref PubMed Scopus (283) Google Scholar). Photoaffinity labeling of rat liver GST 1-1 by glutathionyl S-[4-(succinimidyl)-benzophenone] also results in one subunit being modified (28Wang J. Bauman S. Colman R.F. J. Biol. Chem. 2000; 275: 5493-5503Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Other precedence for binding only 1 mol reagent/mol enzyme dimer comes from work with large conjugation products, such asS-[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathione (29Schramm V.L. McCluskey R. Emig F.A. Litwack G. J. Biol. Chem. 1984; 259: 714-722Abstract Full Text PDF PubMed Google Scholar), and the aflatoxin glutathione conjugate, 8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin, which bind to α class glutathione S-transferases with a stoichiometry of 1 mol/mol dimer (25McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In the case of glutathionylS-[4-(succinimidyl)benzophenone), only one subunit is modified, yet the enzyme is completely inactivated. The modification of one subunit thus can abolish the enzyme activity of both subunits and, because this label does not occupy the nonsubstrate site, the inhibition is probably the result of a subtle conformational change rather than a physical barrier to the binding of the substrate (28Wang J. Bauman S. Colman R.F. J. Biol. Chem. 2000; 275: 5493-5503Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). There is also complete inactivation by the aflatoxin conjugate, although in this case, the bound conjugate extends into the cleft and therefore may be inhibiting completely either because it is blocking access to the active site of the unmodified subunit or because it induces a conformational change (25McHugh T.E. Atkins W.M. Racha J.K. Kunze K.L. Eaton D.L. J. Biol. Chem. 1996; 271: 27470-27474Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In the present case, maximum reaction with 17β-IES results in the loss of only 40% of activity; it is likely that the unmodified subunit retains full activity, whereas the other subunit with modified Cys17 is 80% inactive. Incorporation of 17β-IES on one subunit apparently prevents a second molecule from binding to and reacting with the other subunit, but, in contrast to the previous examples, this does not cause complete inactivation of both subunits. The 17β-IES reacts at the steroid site, which is distinct from the active site, and thus there is still some residual activity in the modified subunit, whereas the catalytic site on the other subunit functions independently and is completely active. These results indicate that the observation of apparent cooperativity between the subunits of glutathione S-transferase depends on the particular binding site that is being examined. A homology model for the rat 1-1 isozyme was generated from the crystal structure of the human glutathione S-transferase 1-1. The reagent was manually docked into the model based on an energy-minimized structure of 17β-estradiol-3,17-disulfate bound to GST 1-1 and the assumptions that the iodoacetoxy group of the 17β-IES must be close to the sulfhydryl group of Cys17 as well as in an orientation to modify only one subunit. The structure shown in Fig.6 meets these requirements. In the proposed model, there is 1 mol of 17β-IES bound in the cleft between the subunits of the enzyme. The reactive iodoacetoxy group is about 3.4 Å from the sulfhydryl group of CysA17. This orientation prevents a second molecule from reacting at Cys17 on subunit B. 17β-IES appears to bind more specifically than does 3β-(iodoacetoxy)dehydroisoandrosterone, which modified both Cys17 and Cys111 (15Barycki J.J. Colman R.F. Arch. Biochem. Biophys. 1997; 345: 16-31Crossref PubMed Scopus (28) Google Scholar). In the case of 3β-IDA, reaction at the two sites were mutually exclusive,i.e. reaction with Cys17 on one subunit excludes binding and reaction with Cys17 on the other subunit. We now propose that the more specific reaction of 17β-IES with only Cys17 is due to an interaction between the sulfate group of 17β-IES and the guanidino group of Arg14; this interaction would orient the reagent within the binding cleft. Based on the model, the charged sulfate group of 17β-IES is about 3.1 Å from the guanidino group of Arg14. In summary, 17β-IES functions as an affinity label of the nonsubstrate steroid site of rat liver glutathioneS-transferase, isozyme 1-1. Upon incubation with 17β-IES, the enzyme loses 40% of its activity, incorporates about 0.5 mol of reagent/enzyme subunit, and is modified only at Cys17. Protection against inactivation by 17β-IES is best provided by steroid sulfates, such as 17β-estradiol-3,17-disulfate, indicating that Cys17 is within the nonsubstrate steroid binding site of the enzyme and that its binding is more specific than that of 3β-IDA because of the interaction of the sulfate group with the side chain of Arg14. Based on analysis of molecular models, this nonsubstrate site is located within the cleft between the two subunits of the enzyme. We thank Dr. Yu-Chu Huang for obtaining the peptide sequences.