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

Melissa A. Vargo and Roberta F. ColmanDagger

From the Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716

Received for publication, September 7, 2000, and in revised form, October 10, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (kobs) exhibits a nonlinear dependence on reagent concentration with KI = 71 µM and kmax = 0.0133 min-1. Complete protection against inactivation is provided by 17beta -estradiol-3,17-disulfate, whereas Delta 5-androstene-3,17-dione and S-methylglutathione have little effect on kobs. 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 Delta 5-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 Cys17 is the only enzymic amino acid modified. Covalent modification of Cys17 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 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-4). 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. Glutathione S-transferases have been found in elevated levels within cancerous tumors and have been implicated in the development of resistance to anti-cancer drugs (5). The cytosolic enzymes are now grouped into seven classes and within a particular class they can exist as either homo- or heterodimers (1). There are crystal structures to represent most of the classes (6-12). Each subunit of the dimer contains a glutathione-binding site and a xenobiotic site that can accommodate a wide variety of compounds.

Isozyme 1-1,2 a member of the alpha  class, efficiently catalyzes the isomerization reaction of Delta 5-androstene-3,17-dione to Delta 4-androstene-3,17-dione, which it binds at the substrate steroid site (13). 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 (14, 15). This site has been proposed to fulfill a transport function (5) or to act in controlling levels of steroids in target organs (16). The nonsubstrate site has a preference for steroid sulfates, which is illustrated by the more potent inhibitory effect of 17beta -estradiol-3,17-disulfate as compared with that of 17beta -estradiol. However, previous work in this laboratory (aimed at locating the nonsubstrate site) used the affinity label 3beta -(iodoacetoxy)dehydroisoandrosterone (3beta -IDA) (shown in Fig. 1), which is structurally related both to substrates of the enzyme, such as Delta 5-androstene-3,17-dione, and to inhibitors of the enzyme, such as Delta 5-androstene-3beta ,17beta -diol disulfate and 17beta -estradiol-3,17-disulfate. The 3beta -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 3beta -IDA is located in the cleft between the subunits (15). Based on the previous data, we have now designed a more specific affinity label for the nonsubstrate steroid site: 17beta -iodoacetoxy-estradiol-3-sulfate (17beta -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 3beta -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 (17). 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 (18).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Steroids that bind to glutathione S-transferase, isozyme 1-1. Delta 5-Androstene-3,17-dione (substrate), 17beta -estradiol-3,17-disulfate (reversible inhibitor), 3beta -(iodoacetoxy)dehydroisoandrosterone (affinity label), and 17beta -iodoacetoxy-estradiol-3-sulfate (affinity label) are shown.



    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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, alpha -chymotrypsin, 17beta -estradiol-3,17-disulfate, 17beta -estradiol-3-sulfate, N,N'-dicyclohexylcarbodiimide, and 1-chloro-2,4-dinitrobenzene were purchased from Sigma. 17beta -Estradiol-17-sulfate and Delta 5-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.

Enzyme Preparation-- Glutathione S-transferase isozyme 1-1 was purified from rat livers using affinity column chromatography on S-hexylglutathione-Sepharose (19). Values of epsilon 270 nm = 22,000 M-1 cm-1 (20) and molecular weight of 25,500 per subunit (2) for GST 1-1 were used to calculate the enzyme concentration.

Synthesis of 17beta -Iodoacetoxy-estradiol-3-sulfate-- 17beta -IES was synthesized from 17beta -estradiol-3-sulfate and iodoacetic acid by procedures based on the method of Pons et al. (21). One molar equivalent of 17beta -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 17beta -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 17beta -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 17beta -IES eluted at ~28 min. For comparison, the starting material, 17beta -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 1810 M-1 cm-1, with the concentration determined from the specific radioactivity.

Enzymatic Assays-- 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 (Delta epsilon  = 9.6 mM-1 cm-1) in 0.1 M potassium phosphate buffer, pH 6.5, at 25 °C according to Habig et al. (22).

Reaction of 17beta -IES with Glutathione S-transferase, Isozyme 1-1-- Glutathione S-transferase (0.2 mg/ml, 7.8 µM enzyme subunits) was incubated in 0.1 M potassium phosphate buffer, pH 7.0, at 37 °C with various concentrations of 17beta -IES. Control enzyme samples were incubated under the same conditions but without 17beta -IES. At various time points, an aliquot was removed from the incubation mixture, diluted, and assayed (30 µl) for residual activity.

Measurement of Incorporation of 17beta -IES into Glutathione S-Transferase-- Glutathione S-transferase (0.2 mg/ml) was incubated with 500 µM [14C]17beta -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 (23). 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 (24). 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 17beta -IES/mol of enzyme subunit.

Preparation and Separation of Proteolytic Digest of Modified Glutathione S-Transferase-- Glutathione S-transferase (0.2 mg/ml) was incubated with 500 µM 14C-labeled 17beta -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 A220, and 1-ml fractions were collected. An aliquot (300 µl) from each fraction was added to 5 ml of Liquiscint to test for radioactivity.

Sequence Determination of Separated Peptides-- 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-- 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 (19) based on the known crystal structure of human liver isozyme 1-1 (1GUH). The structure of 17beta -IES was constructed using the Builder module. Docking of 17beta -IES was done manually based on the energy minimized structure of 17beta -estradiol-3,17-disulfate docked into isozyme 1-1 (15).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inactivation of Rat Liver Glutathione S-transferase 1-1 by 17beta -IES-- Incubation of rat GST 1-1 (0.2 mg/ml, 7.8 µM enzyme subunits), with 300 µM 17beta -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. 2A. After 180 min, excess reagent was removed, and a second addition of 300 µM 17beta -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 17beta -IES concentrations, the data were calculated using 60% as the end point (Fig. 2B). Control enzyme incubated under the same conditions but with no reagent present shows no loss of activity. The kobs for inactivation was calculated from the slope of ln([Et - Einfinity ]/[E0 - Einfinity ]) versus time where Et is the enzyme activity at time t, E0 is the original enzyme activity, and Einfinity is the enzyme activity at long times, which is equal to 0.6 (E0). The reaction obeys pseudo-first order kinetics with a rate constant of 0.0125 min-1 (Fig. 2B).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Inactivation of glutathione S-transferase, isozyme 1-1, by 17beta -IES. A solution of 0.2 mg/ml GST 1-1 was incubated with 300 µM 17beta -IES in 0.1 M potassium phosphate buffer, pH 6.5, at 37 °C. Activity was measured using the substrates 1-chloro-2,4-dinitrobenzene and glutathione, as described under "Experimental Procedures." A, semilog plot of enzyme activity at time t (Et) versus time. B, semilogarithmic plot of [ Et - Einfinity ]/[E0 - Einfinity ] versus time, where E0 is the original enzyme activity, and Einfinity is the enzyme activity at long times, which = 0.6 (E0). The apparent rate constant (kobs) determined from this graph was 0.0125 min-1.

Concentration Dependence of the Rate of Inactivation-- GST 1-1 (0.2 mg/ml, 7.8 µM enzyme subunits) was incubated with 20-300 µM of 17beta -IES as described above, to determine the rate of inactivation at various reagent concentrations (Fig. 3). The apparent rate constant kobs 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 (26). The curve can be described by the equation kobs = kmax/(1 + KI/[17beta -IES]), where KI is the apparent dissociation constant of the enzyme-reagent complex, and kmax is the maximum rate of inactivation at saturating concentrations of the reagent. A least squares fit of the observed data yields KI = 71.4 µM and kmax = 0.0133 min-1.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3.   Concentration dependence of kobs for the inactivation of glutathione S-transferase by 17beta -IES. GST (0.2 mg/ml) was incubated with a range of concentrations of 17beta -IES under the same conditions as Fig. 2. At each concentration kobs was calculated as illustrated in Fig. 2B, with Einfinity  = 0.6 (E0). The points are experimental and the line is the theoretical fit to kobs = kmax/(1 + (KI/[17beta -IES])). A least squares fit of the data yields KI = 71.4 µM and kmax = 0.0133 min-1.

Effect of Ligands on the Inactivation Rate of GST 1-1 by 17beta -IES-- Various ligand analogues were added to the reaction mixture to determine whether they could protect against the inactivation of the enzyme by 100 µM 17beta -IES. The results, given in Table I, are expressed as k+L/k-L, where k+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, yet k+L/k-L does not decrease below 0.37. These results suggest that the target site of 17beta -IES is near the glutathione site but distinct from it. Electrophilic substrates, such as Delta 5-androstene-3,17-dione (Table I, line 4), do not provide any protection. In contrast, including steroid sulfates, such as 17beta -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 (15), the results indicate that 17beta -IES is reacting within this nonsubstrate steroid binding site.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Effects of enzyme ligands on the inactivation of glutathione S-transferase by 300 µM 17beta -IES
The inactivation reaction was conducted at 37 °C in 0.1 M potassium phosphate buffer, pH 6.5.

Incorporation of Radioactive 17beta -IES into GST 1-1-- GST 1-1 (0.2 mg/ml) was incubated with 300 µM [14C]17beta -IES. A time-dependent incorporation of [14C]17beta -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 of 14C-labeled reagent incorporated per mol of enzyme subunit or 1 mol/enzyme dimer at 100% of maximum inactivation.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Incorporation of 14C-labeled 17beta -IES into GST isozyme 1-1, as a function of the percentage of maximum inactivation. Extrapolation to the maximum inactivation of the enzyme reveals an incorporation of about 0.46 mol reagent/mol enzyme subunit.

Isolation and Characterization of Chymotryptic Peptides from 17beta -IES Modified GST 1-1-- 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 17beta -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 Table II. 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 17beta -IES.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Fractionation by HPLC of chymotryptic digest of [14C] 17beta -IES modified gluathione S-transferase.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Representative sequence of modified peptide (Peak I) isolated from the chymotryptic digest of 14C-labeled glutathione S-transferase 1-1, as illustrated by Fig. 5



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

17beta -Iodoacetoxy-estradiol-3-sulfate acts as an affinity label of rat liver glutathione S-transferase isozyme 1-1. Upon incubation of the enzyme with 17beta -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 as S-hexylglutathione, provide more protection than do shorter chain derivatives, like S-methylglutathione, indicating that 17beta -IES is binding in a site close to the glutathione site but not within the site. Electrophilic substrate analogues, such as Delta 5-androstene-3,17-dione, do not offer any protection, demonstrating that 17beta -IES does not bind within the electrophilic substrate site. Steroid sulfates are most effective in protecting against inactivation of GST, 1-1, with 17beta -estradiol-3,17-disulfate providing complete protection. These results indicate that 17beta -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 17beta -IES/enzyme dimer, and Cys17 is the only amino acid that is modified. In previous work, based on the crystal structure of glutathione S-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 (27). Photoaffinity labeling of rat liver GST 1-1 by glutathionyl S-[4-(succinimidyl)-benzophenone] also results in one subunit being modified (28). Other precedence for binding only 1 mol reagent/mol enzyme dimer comes from work with large conjugation products, such as S-[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathione (29), and the aflatoxin glutathione conjugate, 8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin, which bind to alpha  class glutathione S-transferases with a stoichiometry of 1 mol/mol dimer (25).

In the case of glutathionyl S-[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 (28). 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 (25).

In the present case, maximum reaction with 17beta -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 17beta -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 17beta -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 17beta -estradiol-3,17-disulfate bound to GST 1-1 and the assumptions that the iodoacetoxy group of the 17beta -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.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Homology model of rat GST 1-1 complexed with 17beta -IES, constructed as described under "Experimental Procedures." The reactive iodoacetoxy group is about 3.4 Å from the sulfhydryl group of CysA17, and the sulfate group is about 3.1 Å from the guanidino group of Arg14.

In the proposed model, there is 1 mol of 17beta -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. 17beta -IES appears to bind more specifically than does 3beta -(iodoacetoxy)dehydroisoandrosterone, which modified both Cys17 and Cys111 (15). In the case of 3beta -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 17beta -IES with only Cys17 is due to an interaction between the sulfate group of 17beta -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 17beta -IES is about 3.1 Å from the guanidino group of Arg14.

In summary, 17beta -IES functions as an affinity label of the nonsubstrate steroid site of rat liver glutathione S-transferase, isozyme 1-1. Upon incubation with 17beta -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 17beta -IES is best provided by steroid sulfates, such as 17beta -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 3beta -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.


    ACKNOWLEDGEMENT

We thank Dr. Yu-Chu Huang for obtaining the peptide sequences.


    FOOTNOTES

* This work was supported by United States Public Health Service Grants RO1 CA 66561 and T32 GM 08550 (to M. A. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 302-831-2973; Fax: 302-831-6335; E-mail: rfcolman@chem.udel.edu.

Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M008212200

2 Glutathione S-transferase, isozyme 1-1, is designated as the rGSTA1,2 isozyme in the nomenclature of Hayes and Pulford (5).


    ABBREVIATIONS

The abbreviations used are: GST, glutathione S-transferase; 3beta -IDA, 3beta -(iodoacetoxy)dehydroisoandrosterone; 17beta -IES, 17beta -iodoacetoxy-estradiol-3-sulfate; HPLC, high pressure liquid chromatography.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Armstrong, R. N. (1997) Chem. Res. Toxicol. 10, 2-18[CrossRef][Medline] [Order article via Infotrieve]
2. Mannervik, B., and Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 283-337[Medline] [Order article via Infotrieve]
3. Wilce, M. C. J., and Parker, M. W. (1994) Biochim. Biophys. Acta 1205, 1-18[Medline] [Order article via Infotrieve]
4. Pickett, C. B., and Lu, A. Y. H. (1989) Annu. Rev. Biochem. 58, 743-764
5. Hayes, J. D., and Pulford, D. J. (1995) CRC Crit. Rev. Biochem. Mol. Biol. 30, 445-600
6. Ji, X., Zhang, P., Armstrong, R. N., and Gilliland, G. L. (1992) Biochemistry 36, 10169-10184
7. Sinning, 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., and Jones, T. A. (1993) J. Mol. Biol. 232, 192-212[CrossRef][Medline] [Order article via Infotrieve]
8. Reinemer, P., Dirr, H. W., Ladenstein, R., Schaffer, J., Gallay, O., and Huber, R. (1991) EMBO J. 10, 1997-2005[Abstract]
9. Ji, X., Tordova, M., O'Donnell, R., Parsons, J. F., Hayden, J. B., Gilliland, G. L., and Zimniak, P. (1997) Biochemistry 36, 9690-9702[CrossRef][Medline] [Order article via Infotrieve]
10. Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., and Parker, M. W. (1997) Biochemistry 36, 576-585[CrossRef][Medline] [Order article via Infotrieve]
11. Wilce, M. C. J., Board, P. G., Feil, S. C., and Parker, M. W. (1995) EMBO J. 14, 2133-2143[Abstract]
12. Ji, X., Von Rosenvinge, E. C., Johnson, W. W., Tomarev, S. I., Piatigorsky, J., Armstrong, R. N., and Gilliland, G. L. (1995) Biochemistry 34, 5317-5328[Medline] [Order article via Infotrieve]
13. Benson, A. M., Talalay, P., Keen, J. H., and Jakoby, W. B. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 158-162
14. Hu, L., and Colman, R. F. (1997) Biochemistry 36, 1635-1645[CrossRef][Medline] [Order article via Infotrieve]
15. Barycki, J. J., and Colman, R. F. (1997) Arch. Biochem. Biophys. 345, 16-31[CrossRef][Medline] [Order article via Infotrieve]
16. Listowsky, I. (1993) Structure and Function of Glutathione S-transferases , pp. 199-209, CRC Press, Boca Raton, FL
17. Wilchek, M., and Givol, D. (1977) Methods Enzymol. 46, 153-157[Medline] [Order article via Infotrieve]
18. Vargo, M. A., and Colman, R. F. (2000) Biochemistry 39, 1544
19. Wang, J., Barycki, J. J., and Colman, R. F. (1996) Protein Sci. 5, 1032-1042[Abstract/Free Full Text]
20. Katusz, R. M., and Colman, R. F. (1991) Biochemistry 30, 1230-1238
21. Pons, M., Nicolas, J. C., Boussioux, A. M., Descombs, B., and Crastes de Paulet, A. (1973) FEBS Lett. 36, 23-30[CrossRef][Medline] [Order article via Infotrieve]
22. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) J. Biol. Chem. 249, 7130-7139
23. Penefsky, H. S. (1979) Methods Enzymol. 56, 527-530[Medline] [Order article via Infotrieve]
24. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
25. McHugh, T. E., Atkins, W. M., Racha, J. K., Kunze, K. L., and Eaton, D. L. (1996) J. Biol. Chem. 271, 27470-27474[Abstract/Free Full Text]
26. Colman, R. F. (1997) in Protein Function: A Practical Approach, 2nd Ed, Chapter 16, pp. 155-183, Oxford University Press, New York
27. McTigue, M. A., Williams, D. R., and Tainer, J. A. (1995) J. Mol. Biol. 246, 21-27[CrossRef][Medline] [Order article via Infotrieve]
28. Wang, J., Bauman, S., and Colman, R. F. (2000) J. Biol. Chem. 275, 5493-5503[Abstract/Free Full Text]
29. Schramm, V. L., McCluskey, R., Emig, F. A., and Litwack, G. (1984) J. Biol. Chem. 259, 714-722[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.