The Three-dimensional Structure of Cys-47-modified Mouse Liver Glutathione S-Transferase P1-1
CARBOXYMETHYLATION DRAMATICALLY DECREASES THE AFFINITY FOR GLUTATHIONE AND IS ASSOCIATED WITH A LOSS OF ELECTRON DENSITY IN THE alpha B-310B REGION*

M. Cristina VegaDagger , Sinead B. Walsh§, Timothy J. Mantle§, and Miquel CollDagger par

From the Dagger  Departament de Biologia Molecular i Cel·lular, Centre d'Investigació i Desenvolupament-Consell Superior d'Investigacions Científiques, Jordi Girona 18-26, 08034 Barcelona, Spain and the § Department of Biochemistry, Trinity College, Dublin 2, Ireland.

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The three-dimensional structure of mouse liver glutathione S-transferase P1-1 carboxymethylated at Cys-47 and its complex with S-(p-nitrobenzyl)glutathione have been determined by x-ray diffraction analysis. The structure of the modified enzyme described here is the first structural report for a Pi class glutathione S-transferase with no glutathione, glutathione S-conjugate, or inhibitor bound. It shows that part of the active site area, which includes helix alpha B and helix 310B, is disordered. However, the environment of Tyr-7, an essential residue for the catalytic reaction, remains unchanged. The position of the sulfur atom of glutathione is occupied in the ligand-free enzyme by a water molecule that is at H-bond distance from Tyr-7. We do not find any structural evidence for a tyrosinate form, and therefore our results suggest that Tyr-7 is not acting as a general base abstracting the proton from the thiol group of glutathione. The binding of the inhibitor S-(p-nitrobenzyl)-glutathione to the carboxymethylated enzyme results in a partial restructuring of the disordered area. The modification of Cys-47 sterically hinders structural organization of this region, and although it does not prevent glutathione binding, it significantly reduces the affinity. A detailed kinetic study of the modified enzyme indicates that the carboxymethylation increases the Km for glutathione by 3 orders of magnitude, although the enzyme can function efficiently under saturating conditions.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Glutathione S-transferases (GSTs)1 are a widely distributed family of detoxifying enzymes (1). They catalyze the nucleophilic attack of the sulfur atom of the tripeptide glutathione (GSH) on electrophilic groups of a variety of hydrophobic compounds. Six different classes of mammalian soluble GSTs (Alpha, Mu, Pi, Theta, Sigma, and Kappa) have been described, each one with different but overlapping hydrophobic substrate specificity (2-7). The three-dimensional structure of representative enzymes of each class are known (8-17). The different classes show different active site structures, in particular at the hydrophobic subsite (for reviews see Refs. 18 and 19). Despite these differences it is believed that they all share a similar catalytic mechanism where the thiol group of glutathione is activated to thiolate. The reactive ionized S- group attacks the electrophilic group of the hydrophobic substrate leading to a glutathione S-conjugate with increased aqueous solubility (20). How the activation of glutathione is achieved by the enzyme is unclear. The three-dimensional structures of Pi class GSTs complexed with different inhibitors have shown that the only residue of the enzyme in close proximity to the sulfur atom of glutathione is Tyr-7. Until the first GST structure was solved (8), Cys-47 was considered a catalytic residue because its modification inactivated the enzyme (21, 22). The three-dimensional structure, however, revealed that Cys-47 is over 12 Å away from the active pocket. This residue is located in helix 310B at the C terminus of helix alpha B. This helical structure was found to have high temperature factors and exposed hydrophobic residues (12), and the hypothesis was put forward that it could be disordered in the uncomplexed enzyme.

The hypothesis that the "free" enzyme (i.e. with no GSH or GS-conjugates bound) has a structure different from that seen when GSH or GS conjugates are bound has been implicated in a number of studies. We currently have structures for mGST P1-1 with S-hexylglutathione, S-(p-nitrobenzyl)glutathione (12), and GSH2 bound, and in all of these the thiol of Cys-47 is not accessible to solvent. Indeed this appears to be a feature of all class Pi structures solved to date (8, 9, 13). This is important because some authors (23-25) have presented a considerable body of evidence that implicates the availability of cysteine 47 for mixed disulfide formation as a redox sensor. Cystine forms a mixed disulfide with GST P1-1 in agreement with this hypothesis (24). This is attractive as GST P1-1 is almost unique in having a thiol (Cys-47) that appears to be unreactive when GSH is bound to the enzyme but that may be alkylated or oxidized in the absence of GSH (or GS-conjugates). Indeed we have shown that the kinetics of protection against alkylation by GS-conjugates are entirely consistent with such a hypothesis (26). The simplest model to explain this behavior involves at least two conformations for GST P1-1. For the "free" enzyme the thiol of Cys-47 is reactive and exposed to solvent, whereas the binding of GSH or GS-conjugates stabilizes a distinct conformation (seen in the numerous crystal structures solved to date) where the thiol of Cys-47 becomes inaccessible to solvent and thiol reagents. It should be made clear that binding of GS- conjugates does not physically occlude thiol reagents from access to the thiol of Cys-47 because the side chain is in a small hydrophobic pocket on the outer face of the alpha B-310B helices that form the GSH binding site. It has not been possible to crystallize the "free" enzyme for GSTP1-1 to date. We argued that alkylation of cysteine 47 may trap the "free" enzyme in a distinct conformation that permits further analysis and this has proved correct.

Also it has been proposed that Tyr-7 might be in the tyrosinate form in the free enzyme acting as a general base and activating the thiol group of glutathione by proton abstraction (8, 27, 28) when the substrate binds to the active site. However, to date no structure of an unliganded Pi class GST has been reported, and Dirr et al. (18) have reviewed a number of observations inconsistent with the tyrosinate hypothesis.

We have solved the three-dimensional structure of the mouse liver GST P1-1 enzyme modified by iodoacetic acid at Cys-47 to form the carboxymethylated enzyme (referred to as CM-mGST P1-1 from now on) in an unliganded form and with S-(p-nitrobenzyl)glutathione bound at the active site. The carboxymethylated enzyme shows a complete disorder in the alpha B-310B region with a partial destruction of the active site architecture. However, the environment of Tyr-7 remains unchanged in the free enzyme with no indication of an ionized state for this residue. On the other hand the binding of the inhibitor, S-(p-nitrobenzyl)glutathione, results in a limited restructuring of the disordered area. The results presented here provide a structural explanation for the effect of the chemical modification of Cys-47 and suggest that Tyr-7 is not acting as a general base. It is presently unclear whether carboxymethylation is locking the enzyme in a conformer of the native enzyme, and further work is required to investigate this.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Modified mouse liver GST P1-1 was prepared by incubating 18 mg of the native enzyme (26) with 200 mM iodoacetic acid in 100 mM sodium phosphate, pH 8, in a final volume of 10 ml for 40 min and then removing excess iodoacetic acid by gel filtering on Sephadex G-25 equilibrated in 10 mM sodium phosphate, pH 7. The modified enzyme does not bind to glutathione-Sepharose, so to ensure that any residual native enzyme was removed, the modified preparation was passed through this affinity resin, and the protein that eluted in the wash through was pooled, concentrated using an Amicon filter, and stored at -20 °C. This material, homogenous by the criteria of SDS/polyacrylamide gel electrophoresis and exhibiting a pI of 8.2 (29) exhibits a marked increase in the KmGSH (see below).

Suitable crystals of CM-mGST P1-1 were grown by vapor diffusion at room temperature from hanging drops prepared by mixing 3 µl of protein solution (7 mg/ml) and 3 µl of precipitant solution containing 33 mM Hepes buffer, pH 7, 64 mM CaCl2, and 8.3% PEG 4000. The drops were equilibrated against a reservoir of 19% PEG 4000, 0.15 M CaCl2, 75 mM Hepes buffer. Long prismatic crystals appeared after 1 week. Three-dimensional diffraction data were collected up to 1.9 Å resolution, at 4 °C, using the X11 station, the European Molecular Biology Laboratory at the Deutsches Elektronensynchrotron (lambda  = 0.937 Å). Because of the intensity decay three crystals of approximate size 0.7 × 0.2 × 0.2 mm were necessary to collect a full data set. Images were taken in one-degree rotation steps with the detector set at 165-mm distance. The crystals are tetragonal space group I4, with unit cell dimensions a = b = 132.5 Å and c = 63.2 Å, and have one dimer/asymmetric unit. The data were processed with the XDS package (30) and merged together with the program PROTEIN (31). Intensity data statistics are given in Table I.

                              
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Table I
Crystal parameters and data collection statistics

Crystals of CM-mGST P1-1 complexed with S-(p-nitrobenzyl)glutathione were grown at room temperature from hanging drops. The best crystals appeared by mixing 3 µl of protein solution (7 mg/ml), 1 µl of 20% PEG 8000 in 0.1 M MOPS buffer, pH 6.4, and 0.2 M of ammonium sulfate and 2 µl of inhibitor saturated solution in 0.1 M MES buffer at pH 7.5. The drops were equilibrated against 10% PEG 8000, 50 mM MOPS buffer. After 10 days needle-like crystals grew to a size of 0.6 × 0.2 × 0.1 mm. The crystals diffracted weakly on a rotating anode. However, a 3 Å data set could be collected at the BW7B station, the European Molecular Biology Laboratory at the Deutsches Elektronensynchrotron (lambda  = 0.869 Å), at 4 °C. The crystals are orthorhombic space group P212121 and with cell dimensions a = 62.45, b = 132.69, and c = 214.47 Å and have three protein dimers in the asymmetric unit. The data were processed with DENZO (32). Intensity data statistics are given in Table I.

The coordinates from the mGST P1-1 enzyme as in the S-(p-nitrobenzyl)glutathione complex structure (Protein Data Bank code 1glq) (12) were used as a starting model for the determination of both structures by molecular replacement with AMoRe (33). The solution for the unliganded form was found using two independent monomers as initial model and a resolution range of 12-4 Å. After the rigid body refinement routine of AMoRe, the correlation coefficient (for Fobs) was 0.75, and the R-factor was 0.303. A combined x-ray and energy minimization refinement followed with XPLOR (34), including cycles of simulated annealing. In the refinement process 10% of the reflections were set aside for the R-free calculation (35). Fourier electron density maps were calculated at 3 Å resolution with coefficients 2Fo - Fc and Fo - Fc and displayed using TURBO (36). Most of the molecule fitted correctly in the electron density except for residues 35-51, where the electron density was absent in the maps contoured at 1 and 2 sigma , respectively. No protein atoms could be located in this area. Further refinement cycles followed adding solvent molecules and slowly increasing the resolution up to 2.0 Å. Atomic isotropic B factors were refined with restraints imposed on atoms directly bonded. At this stage the R-cryst was 0.197, and the R-free was 0.236. A final run of positional and B-factor refinement was carried out including all data consisting in 26183 reflections (Fobs > 2sigma Fobs) in the resolution range 8.0-2.0 Å. This resulted in a final R-factor of 0.195 (Table II). The final structure consists of 385 protein residues of 418 amino acids of the chemical sequence and 150 solvent molecules.

                              
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Table II
Refinement statistics

To solve the structure of CM-mGST P1-1 complexed with S-(p-nitrobenzyl)glutathione, three dimers of mGST P1-1 were used as initial model. After the rigid body refinement routine of AMoRe, a correlation coefficient (for Fobs) of 0.53 and an R-factor of 43.7% were obtained for the correct solution. Refinement of the structure followed with MAIN (37), omitting the area corresponding to the alpha -helix B that was disordered in the unliganded structure. Initially, the noncrystallographic symmetry (NCS) operators relating the two monomers in each dimer and the three dimers of the asymmetric unit were determined from the molecular model. With these symmetry operators and masks build around the molecules, the map was subject to 6-fold density averaging and solvent flattening. Iterative cycles of density averaging and refinement of the NCS operators followed, maximizing the correlation among the six monomers. After this procedure, inspection of the active sites in 3 Å resolution Fourier maps showed clear electron density corresponding to the inhibitor molecules. The inhibitor S-(p-nitrobenzyl)glutathione was fitted in the electronic density (Fig. 1). Also part of the disordered area in the unliganded enzyme now showed clear density, and the protein model was build accordingly. Further refinement of the model using NCS restraints was performed with MAIN giving an R-factor of 0.27. Simulated annealing and positional refinement with XPLOR followed, setting aside 5% of the reflections for R-free calculation. After NCS-restrained B-factor refinement, the R-cryst was 0.246, and the R-free 0.277 (Table II). Because of the limited resolution of this data set, we decided to conclude the refinement process at this point, not including solvent molecules and not breaking the NCS restraints to maintain a reasonable parameter/data ratio. The final structure consists of three protein dimers of 398 residues each of 418 amino acids of the chemical sequence and 6 inhibitor molecules. Atomic coordinates and structure factors for both the unliganded and complexed CM-mGST-pi structures have been deposited with the Brookhaven Protein Data Bank.


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Fig. 1.   Fo - Fc electron density map at the active site area of the CM-mGST P1-1· S-(p-nitrobenzyl)glutathione complex calculated before introducing the inhibitor molecule. The S-(p-nitrobenzyl)glutathione has been fitted in the density.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Structure of the Cys-47-carboxymethylated Enzyme-- The global fold of the unliganded CM-mGST P1-1, excluding residues 34-51, is similar to that of the native enzyme when complexed with glutathione adducts. The rms deviation in the Calpha positions is 0.33 Å between molecule A of the present structure and molecule A in the S-(p-nitrobenzyl)glutathione complex structure. Other comparisons show similar values. The rms difference between monomers in the present structure is 0.107 Å.

The main difference with the native complexed structure is at helix alpha B and the following 310 helix. These helices form the external wall of the active site (Fig. 2, in red). Helix alpha B runs in the native structure from Thr-34 to Gln-40. It is followed by a bend formed by Gly-41 and Leu-42 and helix 310B, which runs from Leu-43 to Cys-47. After the 310 helix a beta -turn, including residues Cys-47 to Gly-50, is observed. There are several residues in this area that are part of the active site. The side chains of Trp-38, Lys-44, and Gln-51 and the NH group of Leu-52 have H-bond interactions with the glutathione substrate (12), and therefore they belong to the G-subsite. On the other hand Ile-35 is part of the hydrophobic or H-subsite. In the iodoacetic acid modified enzyme this substructure is completely disordered from Ile-35 to Gln-51 with no density in the Fourier maps (Fig. 3A). The 17-residue disordered area contrasts with the good definition of the rest of the structure. A comparison of the native and the Cys-47-derivatized structures in this area can be observed in Fig. 3B.


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Fig. 2.   Calpha trace of the Cys-47-carboxymethylated mGST P1-1 dimer. The inhibitor S-(p-nitrobenzyl)glutathione is represented by pink (aromatic ring) and blue balls. The disordered zone in the unliganded CM-mGST P1-1 is shown in orange. It includes the external helices alpha B and 310B and affects mainly the G-subsite.


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Fig. 3.   A, 2Fo - Fc Fourier map of the disordered zone. Note the lack of electronic density from Ile-35 to Gln-51. B, superposition of structures at the Ile-35-Gln-51 area; native GST P1-1 in the complex with S-(p-nitrobenzyl)glutathione (blue) (Ref. 12), unliganded CM-mGST P1-1 (yellow), and liganded CM-mGST P1-1 (orange).

Apart from the disorder of the helix B zone no changes at all are observed affecting other residues. In particular Tyr-7 remains at the same position as in the complexes and with a similar environment except for a water molecule (W0) that now occupies the position of the sulfur atom of glutathione in the complexes and is at H-bond distance from the hydroxyl oxygen of the Tyr-7 (Figs. 4 and 5B). Therefore, the Tyr-7 hydroxyl group interacts only with the NH amide of Arg-13 (2.9-2.8Å) and the aforementioned water molecule, W0. The side chain of Arg-13 has not changed its position and maintains its salt bridge and double hydrogen bond with Glu-97. Other residues from the G-subsite not affected by the disorder are Gln-64, Ser-65, and Asp-98 (from the other monomer). At the H-subsite Phe-8, Val-10, and Gly-205 remain at the same positions as in the native complexes. It is remarkable that Phe-8 stays in place with its side chain unperturbed.


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Fig. 4.   2Fo - Fc (blue) and Fo - Fc (red) Fourier maps in the vicinity of Tyr-7 before assigning the residual density (superimposed red and blue globule) to water molecule W0.


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Fig. 5.   Active sites in two different structures. A, native mGST P1-1 complexed with S-(p-nitrobenzyl)glutathione (Protein Data Bank code 1glq) (12). B, unliganded CM-mGST P1-1. Water molecules W1, W2, and W3 are preserved in both the liganded native and in the unliganded carboxymethylated enzymes. W0 occupies in the free enzyme the location of the thiol group of glutathione.

Fig. 5 shows the water structure at the active site. In the native complexed structure a row of three water molecules, W1, W2, and W3, are located close to the sulfur atom, with W1 at H-bond distance from it and W3 H-bonded to Tyr-108 (Fig. 5A). In the unliganded Cys-47-modified structure the 3 water molecules are present (Fig. 5B). W1 is now H-bonded to W0, which occupies the GSH sulfur position. The active site is filled by three more water molecules at the H-subsite, W4, W5, and W6, with W4 H-bonded to the hydroxyl group of Tyr-108. W4 is also present in the glutathione-GST complex at the H-subsite.2

Structure of the Cys-47-carboxymethylated Enzyme Complexed with S-(p-Nitrobenzyl)glutathione-- The global fold of the CM-mGST P1-1 in the complexed enzyme is again similar to the native complexed enzyme. For example, the rms deviation in the Calpha positions, excluding residues 38-48, is 0.37 Å between molecule A of the present structure and molecule A in the S-(p-nitrobenzyl)glutathione complex structure (12). The S-(p-nitrobenzyl)glutathione is present at the active site (Fig. 1). When the structure of CM-mGST P1-1 (Fig. 3B) is compared with the structure obtained in the presence of S-(p-nitrobenzyl)glutathione, it is obvious that the disordered helix B region is partially restructured on binding ligand. For example, residues Ile-35, Asp-36, Thr-37, and Trp-38 are now in well defined electron density. Cys-47, Leu-48, Tyr-49, Gly-50, and Gln-51 are also in density.

Thus, one residue of the H-subsite (Ile-35) and two residues of the G-subsite (Trp-38 and Gln-51) have been stabilized by the binding of the product analogue S-(p-nitrobenzyl)glutathione and are now interacting with it. However Lys-44, another G-subsite residue, remains disordered, and this would account for a weaker binding of the glutathione moiety when compared with the native protein.

The position of all the active site residues except for Lys-44 is almost identical in the native and derivative complexes, although the Trp-38 side chain shows a small rotation around its chi 2 side chain torsion angle (Fig. 3B). Both the glutathione and p-nitrobenzyl moieties of the inhibitor show the same conformation as in the native mGST P1-1 complex with S-(p-nitrobenzyl)glutathione. Tyr-7 is again the only residue close to the sulfur atom of glutathione at 2.9-3.4 Å distance.

Kinetic Analysis of the Cys-47-carboxymethylated Enzyme-- During our initial purification of the carboxymethylated protein we confirmed the report of Tamai et al. (21) that the thiol-modified enzyme (CM-mGST P1-1) does not bind to S-hexylglutathione-Sepharose or to glutathione-Sepharose. However, we observed very low levels of 1-chloro-2,4-dinitrobenzene-conjugating activity in the wash-through fraction and, in preliminary work, could find no evidence for contamination with unmodified protein (29) consistent with the hypothesis that the modified enzyme retained some residual activity. By monitoring the wash through material from the affinity column at far higher concentrations of glutathione (20-100 mM) than we would normally use (1 mM), it is possible to detect significant 1-chloro-2,4-dinitrobenzene-conjugating activity in the nonbound fraction. Detailed kinetic studies indicate that the CM reaction increases the Km for glutathione from 150 µM to approximately 100 mM (Fig. 6). We have confirmed this apparent decrease in affinity for GSH by monitoring the GSH-dependent quenching of protein fluorescence when values for KDGSH for the native and CM-modified protein are 150 µM and 80 mM, respectively. The Km for the second substrate 1-chloro-2,4-dinitrobenzene is hardly affected (1 mM native enzyme and 2 mM for the CM-modified enzyme). The turnover number for the CM-modified enzyme is 30 s-1, which is close to the value reported by Phillips (38) for the native enzyme of 51 s-1. It thus appears that the CM modification simply increases the Km for glutathione by almost 3 orders of magnitude but that under saturating conditions the enzyme turns over almost as efficiently as the native enzyme. This kinetic data is entirely consistent with the partial destructuring of the glutathione binding site but with no rearrangement around the catalytic tyrosine. Interestingly the addition of a hydrophobic moiety (p-nitrobenzyl) to the glutathione sulfur atom results in tight binding to CM-mGST-pi that is similar (38 µM) to the value obtained for the native enzyme (12 µM). Again this is in accord with the structural data where the hydrophobic moiety interacts with the H-site, and for the CM-mGST-pi enzyme the disordered helix B region is partially restructured around the glutathione element.


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Fig. 6.   Initial rate kinetics for the native (bullet ) and carboxymethylated (open circle ) mGST P1-1. The experiments were conducted at 30 °C in 100 mM sodium phosphate buffer at pH 6.7 by varying the concentration of glutathione at a fixed concentration of 1-chloro-2,4-dinitrobenzene (1 mM). The inset shows an extended data set for CM-mGST-pi . The solid line represents a fit to a rectangular hyperbola using the nonlinear, quasi-Newton method in Mac Curve FIT.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cys-47 was believed to be an essential catalytic residue because of its high reactivity and because its chemical modification affects the enzyme activity. Surprisingly the first Pi class GST structure solved (8) showed that this residue was removed about 12 Å from the active site. The structure of the Cys-47-carboxymethylated mGST P1-1 proves that the effect of the chemical modification is a loss of electron density in the helix alpha B and 310B region, a 17-residue stretch including important binding site residues. The loss of electron density may be due to high thermal motions or oscillations between a number of conformers. The side chain of Cys-47 is buried by the side chains of Leu-43, Thr-46, Lys-53, and Tyr-63 in the native complexes and is essentially inaccessible to solvent (see also Ref. 18). In addition the stability of the alpha B-310B substructure must be marginal with exposed hydrophobic residues like Leu-42. The addition of a carboxymethyl group at the Sgamma atom of Cys-47 would cause severe contacts with the surrounding side chains, in particular with Leu-43, and prevent the correct folding of this area.

However, our kinetic studies demonstrate that the carboxymethylated enzyme is still active with a kcat comparable with that of the native protein. Simply the carboxymethylation of Cys-47 increases the Km for glutathione by 3 orders of magnitude. Under saturating conditions the modified enzyme turns over almost as efficiently as the native one. In agreement with this, the complex of CM-mGST-pi with S-(p-nitrobenzyl)glutathione proves that a product analog can bind to the modified enzyme and that this binding partially organizes the active site except for Lys-44. Inhibitor binding results in the organization of 6-10 residues out of 17, but the ligand binding is unable to fully organize the area, most probably because of the steric hindrance of CM-Cys-47 with Leu-43.

The carboxymethylation of Cys-47 even though affecting residues implied in the substrate binding does not affect residues close to the glutathione sulfur site like Tyr-7 or Tyr-108, which might be somehow involved in catalysis (17, 39-41). Neither does it affect the water structure of the pocket where W1, W2, and W3 (Fig. 5) maintain their positions, although extra water molecules appear to fill the space freed by the absence of the substrate. Again this agrees with the turnover capability of the CM-modified enzyme because the appropriate environment around the glutathione thiol or thiolate group is preserved.

Furthermore in the unliganded CM-mGST P1-1 we do not find any evidence that Tyr-7 is in the tyrosinate form. There is no positively charged residue to stabilize a Tyr-O- ion in the vicinity for CM-mGST P1-1, and this is also the case for the native enzyme complexed with glutathione adducts. On the contrary the hydroxyl group of Tyr-7 seems to be protonated, accepting a hydrogen bond from the main chain NH group of Arg-13 and giving a hydrogen bond to a well defined water molecule, W0, that occupies the thiol position in the absence of glutathione (Fig. 5, A and B). W0 is obviously displaced when the substrate glutathione binds, and the H-bond acceptor would then be the glutathione thiol(ate). Our results do not support the hypothesis of a tyrosinate residue acting as a general base and accepting a proton from the glutathione thiol. A similar conclusion has been reported for the human Alpha class GST A1-1 (16). Its role must be just stabilizing the thiolate by hydrogen bonding and positioning it in the right orientation for the nucleophilic attack on the hydrophobic substrate. Because there is strong evidence that the glutathione molecule is in the thiolate form when bound to the enzyme, the most feasible hypothesis is that one of the water molecules at the active site accepts the proton (42). For GST A1-1 Widersten et al. (43) have suggested that a water molecule could relay the thiol proton to the alpha -carboxylate group of glutathione.

The present work does not completely solve the question of whether substrate binding induces conformational changes in native mGST P1-1. In this respect there are two possible scenarios: (i) The first scenario is that unliganded native mGST P1-1 has a disorder similar to the one observed in the free CM-mGST P1-1 enzyme. This disorder affects residues that are involved in binding of glutathione but are not directly implicated in catalysis. The binding of the glutathione substrate would then organize the alpha B-310B area. The H-subsite would be substantially the same in the free enzyme, except for Ile-35, and, therefore, the hydrophobic substrate would readily bind. Ile-35 would close the lipophilic crevice at one side, once the substrate enters. The difficulties in crystallizing the native enzyme in the absence of GSH or GS conjugates seem to indicate some flexibility or disorder, although this argument does not extend to the free CM-mGST P1-1, which was indeed crystallized in spite of having a 17-residue portion of the polypeptide chain disordered. (ii) The alternative scenario consists of a native mGST P1-1 perfectly structured in its free form. The structure of Schistosoma japonica GST, which is different from the mammalian enzymes but related to the Mu class, shows that the active site is unchanged when comparing the ligand-free and the ligand-bound enzyme (15). In the alpha  class an unaltered G-subsite is observed in the absence of ligand, although the distinct C-terminal alpha -helix of this class, which covers the H-subsite when the hydrophobic substrate is bound, appears to be disordered in the unliganded form (16).

For mGST P1-1, with the present data, neither of the two scenarios can be ruled out. However, we note that neither of them implies any rearrangement of residues in the surroundings of Tyr-7 or Tyr-108. Only water molecules that fill the ligand-free active site, some of them H-bonded to these two tyrosine residues, would be expelled by the binding of the substrates.

    ACKNOWLEDGEMENTS

We thank Dusan Turk for setting up the NCS protocol in MAIN, Isabel García-Sáez for help and discussion, and David Sheehan for helpful comments.

    FOOTNOTES

* This work was supported by Ministerio de Educación y Ciencia Grant PB95-0224 and by funds from the Center de Referència en Biotecnologia (Generalitat de Catalunya). Data collection at the European Molecular Biology Laboratory Outstation at the Deutsches Elektronensynchrotron was supported by the European Union Large Installations Project CHGECT93-0040.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.

The atomic coordinates and structure factors (codes 1BAY and R1BAYSF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Supported by a Wellcome Toxicology Studentship.

par To whom correspondence should be addressed. Tel.: 34-3-4006149; Fax: 34-3-2045904; E-mail: mcccri{at}cid.csic.es.

1 The abbreviations used are: GST, glutathione S-transferase; GSH, glutathione; GS-conjugate, glutathione S-conjugate; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; mGST, mouse GST; NCS, noncrystallographic symmetry; CM, carboxymethylation; PEG, polyethylene glycol.

2 A. Párraga, I. García-Sáez, S. B. Walsh, T. J. Mantle, and M. Coll, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Hayes, J. D., Pickett, C. B., and Mantle, T. J. (1990) in Glutathione S-Transferases and Drug Resistance (Hayes, J. D., Pickett, C. B., and Mantle, T. J., eds), pp. 3-15, Taylor & Francis, New York
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