Glutathione Transferase A1-1: Catalytic Importance of Arginine 15

Jan 7, 2010 - Computational Evidence for the Detoxifying Mechanism of Epsilon Class ... Glutathione Transferase Classes Alpha, Pi, and Mu: GSH Activat...
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Glutathione Transferase A1-1: Catalytic Importance of Arginine 15 Daniel F. A. R. Dourado,† Pedro Alexandrino Fernandes,† Bengt Mannervik,‡ and Maria Joa˜o Ramos*,† REQUIMTE/Departamento de Quı´mica Faculdade de Cieˆncias, UniVersidade do Porto Rua do Campo Alegre, 687, 4169-007 Porto, Portugal, and Department of Biochemistry and Organic Chemistry, Uppsala UniVersity, BMC Box 576, SE-75123 Uppsala, Sweden ReceiVed: August 26, 2009; ReVised Manuscript ReceiVed: NoVember 10, 2009

Glutathione transferases (GSTs) are fundamental enzymes of the cell detoxification system. They catalyze the nucleophilic attack of glutathione (GSH) on electrophilic substrates to produce less toxic compounds. The resulting substrate can then be recognized by ATP-dependent transmembrane pumps and consequently expelled from the cell. Despite all the existing studies on GSTs, many aspects of the catalytic events are still poorly understood. Recently, using as a model the GSTA1-1 enzyme, we proposed a GSH activation mechanism. Resorting to the density functional theory (DFT), we demonstrated that a water molecule could assist a proton transfer between the GSH thiol and R-carboxylic groups, after an initial conformational rearrangement of GSH, as evidenced by potential of mean force calculations. In this work to elucidate the catalytic role of Arg15, a strictly conserved active site residue in class alpha GSTs, we analyzed the activation energy barrier and structural details associated with the GSTA1-1 mutants R15A, R15Rε,η-c (an Arg residue with the ε,η-nitrogens substituted by carbons), and R15Rneutral (a neutral Arg residue due to the a addition of a hydride in the ζ-carbon). A similar mechanism to the one used in our GSH activation proposal was implemented. Introduction Since the early 1960s glutathione transferases (GSTs) have been known as xenobiotic/endobiotic detoxification enzymes. In fact, they are described as the most important enzymes involved in the metabolism of electrophilic compounds. Cytosolic classes R, π, and µ are the most extensively studied GSTs.1-50 Structurally, they are formed either by identical monomers or by different monomers within the same class of isoenzymes. Each monomer has two active sites, a G-site and an H-site. The former, conserved across the different GSTs, is highly specific for glutathione (GSH) and is only completed after dimerization, since it is located in a cleft between the N-terminal domain of one subunit and the C-terminal domain of the other. The H-site is found mainly in the C-terminal domain and its structure varies widely between GSTs, allowing for the binding of a vast spectrum of electrophilic toxic compounds. GST catalysis involves the nucleophilic addition of the sulfur thiolate of GSH to a wide range of electrophilic compounds (R-X), building up a less toxic and more soluble conjugate (GSR), which can be removed from the cell. The product (GSR) release, controlled by the C-terminal domain, is the catalytic rate-limiting step for fast substrates.10,50 GSH + R-X(electrophilic substrate) f GSR + H+ + X-

When GSH binds to the G-site, the pKa of the thiol group drops from 9.1 to about 6.2-6.6 pH units,13,21 promoting its deprotonation. The influence of the GSH glutamyl R-carboxylate and the G-site water molecules in this step has been re* To whom correspondence should be addressed. E-mail: mjramos@ fc.up.pt. † Universidade do Porto Rua do Campo Alegre. ‡ Uppsala University.

ported,9,13,22,23,25,26,51,52 but up until recently the nature of the residue that receives the proton from the thiol group was still a matter of debate. We proposed a GSH activation mechanism consistent with the known data.53 Our studies with GSTA1-1 have shown that a water molecule is able to promote/mediate a proton transfer between GSH thiol and R-carboxylic groups with an activation energy of 13.39 kcal · mol-1, following a first conformational rearrangement of GSH (∆Gconf ) -1.62 kcal · mol-1). This energy barrier is consistent with the experimental kinetics for the GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB) (kcat ) 88 ( 3 s-1, ∆qG° ) 15.06 kcal · mol-1 9), a common electrophilic substrate for members of the cytosolic classes R, π, and µ of GST. In another study with the GSTA1-1 enzyme we confirmed the existence of water coordination spheres around the GSH thiol and the glutamate R-carboxylate. We have also shown that for GSTA1-1 residue-68 mutants known for having a low catalytical efficiency, dramatic changes in water arrangement around these GSH groups are observed, further emphasizing the importance of water molecules as promoters of GSH activation.49 Figure 1 shows the G-site active center catalytic residues. On the lefthand side we can see the protonated Tyr9, which acts as a hydrogen bond donor, stabilizing the deprotonated form of the GSH cysteine thiol.21 Substitution of this residue by a phenylalanine results in a vast decrease on catalysis.17-19 On the righthand side it is possible to observe that the GSH glutamyl carboxylic oxygen atoms are hydrogen bonded with Thr68 main chain and side chain, while the GSH glutamyl amino group is hydrogen bonded with the Gln67 side chain. The substitution of a hydroxyl to a methyl group in the T68 V mutation and consequent loss of Thr68 side chain interaction with GSH leads to a reduction in the kcat of 3-6 fold and a KMGSH increase of ∼2-fold.9 In a recent work, we have shown that the loss of residues 67-68 interactions leads to a different active center

10.1021/jp908251z  2010 American Chemical Society Published on Web 01/07/2010

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Figure 1. G-site model representation of the enzymes studied (wild type, R15A, R15Rε,η-c, and R15Rneutral). Residue15 and its intermolecular interactions are highlighted. Distance (δ) between atoms A and B, taken as the reaction coordinate in the PMF calculation, is also shown.

arrangement of GSH and to significant changes in the water arrangement around the GSH thiol and glutamate R-carboxylate groups.49 In the middle of the G-site active center residue Arg15 appears, a highly conserved residue in alpha GSTs, involved in a salt bridge with Glu104. The precise role of Arg15 remains poorly understood. In the R15A and R15H mutants, the specific activity suffers a 200- and 400-fold decrease, respectively, when compared with the wild-type enzyme.21 It should be noted that these mutations have shown an activity even lower than the important Y9F mutant. On the other hand, the R15L mutation has resulted in an activity only 3-fold lower than the wild-type enzyme. Here, by studying the Arg15 intermolecular interactions and steric effects, we propose to clarify the role of this conserved residue for GSH activation. A theoretical study of the activation energy barrier of the following residue 15 mutants was performed: R15A, R15Rε,η-c (an Arg residue with the ε,ηnitrogens substituted by carbons), and R15Rneutral (a neutral Arg residue due to addition of a hydride in the ζ-carbon). Using the approach of theoretical chemistry, we are able to study unnatural amino acid systems, such as the mutants R15Rneutral and R15Rε,η-c, which can provide fundamental insights not accessible to experimentalists. Figure 1 displays the mutants subjected to computational analysis. In the R15A mutant all the side-chain intermolecular interactions cease to exist. The study of the unnatural mutant R15Rε,η-c allows to distinguish

unequivocally the importance of the Arg15 side chain volume by eliminating the nitrogen atoms and consequently its hydrogen bonds and strong ion-dipole interactions. With the study of the mutant R15Rneutral, we can understand the role that the positive charge of Arg15 plays by maintaining the side-chain volume and the nitrogen hydrogen bonds while eliminating its ion-dipole interactions. Methods In subsection I, Conformational Rearrangement of GSH, we have studied the free energy associated to the GSH conformational rearrangement that is needed for a water molecule to interact simultaneously with both the thiol and the glutamyl R-carboxylate groups. For that purpose, we have calculated the potential of mean force (PMF) using the umbrella sampling method. This calculation was performed at the molecular mechanics (MM) level for several reasons: (a) no bond-making or bond-breaking processes were occurring and therefore a quantum mechanics (QM) methodology was not strictly necessary; (b) steric strain plays a significant role in conformational rearrangements and such strain is better captured by the full enzyme environment with its mechanical tensions explicitly considered; and (c) the use of a MM methodology allows the sampling of the conformational space. In subsection II, Proton Transfer Study, we started from the final PMF-generated structure and calculated the energy involved

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Figure 2. Water-assisted proton transfer mechanism. (A) Calculated Gibbs energy changes vs SH-COO- distances. The curve represents the sum of the entire data obtained from the PMF forward and backward processes. On the right-hand side, a detailed view of the GSTA1-1 complex with GSH is represented, highlighting the reaction coordinate. (B) Wild-type and mutant enzymes’ water-assisted proton transfer Gibbs energies of the three stationary points: reagent (R), transition state (TS), and product (P). Energies calculated with DFT, functional B3LYP, and basis set 6-311++G(2d,2p). On the right-hand side the G-site model is represented along with distance σ, decreased at each scan point.

in the intramolecular proton transfer on the wild-type and the mutant enzymes. Similar G-site active center models were built for the wild-type enzyme and the residue-15 mutant enzymes. We then performed scans of the water proton approach to the most suitable GSH glutamate R-carboxylate oxygen and observed the simultaneous thiol group proton transfer to the water molecule. From the scan, structures of the three stationary points (reagents, transition state, and product) were obtained and the proton transfer activation energy, ∆G, was calculated. The full description of subsections I and II follows. I. Conformational Rearrangement of GSH. Molecular Dynamics. The crystallographic structure of the wild-type GSTA1-1 complexed with GSH was obtained in the Protein Data Bank,54 code 1PKW. A water molecule was later added and placed between the GSH thiol and the glutamyl R-carboxylate groups. The γ-glutamyl group of GSH had to be parametrized as there are no parameters in the AMBER99 force field for this species.55,56 The dihedrals, angles, bonds, and van der Waals parameters were based in the AMBER99 force field. Atomic point charges were calculated with the Gaussian software package, following the methodology used in the AMBER99 force field by fitting the HF/6-31G* generated electrostatic potential to atomic point charges using the ESP (RESP) algorithm. All the molecular dynamics simulations and subsequent analyses were carried out using the Gromacs software package conjugated with the AMBER99 force field.55-58 The enzyme models were solvated with ∼17 000 single-point

charge waters (SPC)59 and then subjected to 100 steps of steepest descent energy minimization to remove bad contacts between the solvent and the protein. Subsequently, the system was equilibrated for 200 ps, maintaining the protein atoms restrained by weak harmonic constraints to allow for the structural relaxation of the water models. Fourteen (7 + 7) production simulations of 150 ps were performed with a time step of 0.002 ps. The coordinates and velocities were saved at 1 ps intervals. In all simulations, periodic boundary conditions were used. The temperature and pressure were maintained constant using the Berendsen temperature coupling and pressure coupling (parameters: τT ) 0.1 ps, Tref ) 300 K, Pref ) 1 bar).60 The particle mesh Ewald (PME)61 method was applied to compute electrostatic interactions with a cutoff of 1.0 nm. In terms of van der Waals interactions, a twin range cutoffs with neighbor list cutoff 1.0 and van der Waals cutoff of 1.0 was used. Potential of Mean Force Calculations. The above-mentioned simulations were performed in order to calculate the PMF associated with the approach of the thiol group to the R-carboxylic group, as in the beginning of the simulation both groups are still too distant for an efficient proton transfer. The PMF represents the Gibbs energy change, ∆G, as a function of a coordinate of the system. In our study, the distance (δ) between atoms A and B was taken as the reaction coordinate (Figure 1). It should be also noted that the position of the water molecule was constrained in the reaction coordinate pathway. The PMF calculation was performed following the umbrella sampling

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Figure 3. Reagents for the water-assisted proton transfer: (1) wild type, (2) R15A mutant, (3) R15Rneutral mutant, and (4) R15Rε,η-c mutant. Relevant distances (nm) are shown.

method.62 In the umbrella sampling method, a series of simulation windows are performed along a reaction coordinate and each window is restrained by imposing a harmonic umbrella biasing potential U′(δ)

U(δ) ) 1/2κ(δ - δ0)2 in which κ is the force constant. Distance δ between the hydrogen (atom A) and oxygen (atom B) atoms (Figure 1) was steadily decreased in each window by 0.04 Å. After the last δ value, the backward process was also performed. The force constant was calibrated in order to allow an overlapping of the windows along the reaction coordinate (κ ) 50-125 kcal · mol-1). A total of seven forward and seven backward 150 ps windows were sampled, resulting in an overall 2100 ps production simulation. The unbiased δ probability distribution, in both forward and backward directions, was used to calculate the ∆G associated with the approach of the thiol group to the glutamyl R-carboxylate by the constant temperature weighted histogram analysis method (WHAM).63 The WHAM method allows the calculation of the PMF by computing the unbiased distribution function as a weighted sum over the individual biased distributions of each window. II. Proton Transfer Study. Starting from the final structure of the PMF calculation, we built G-site models that only differ in residue 15 (Figure 1). These models have all atoms critical

to catalysis, including the water molecule positioned between the thiol and the glutamyl R-carboxylate groups (129-143 atoms). In order to safeguard the main structure of the G-site active center the R-carbons were fixed (six constraints) during the whole proton transfer study. It should be also mentioned that the GSH glutamate R-amino group was included in the deprotonated state, since it is at the molecule|vacuum interface, has no other residue nearby to compensate its charge and is not directly related with the reaction coordinate. In order to optimize the computation time, we have resorted to the ONIOM hybrid method64-66 that allows a division of the model in different theoretical levels. This method has recently been used in the catalytic study of several enzymes with excellent results.53,67-72 The high layer includes GSH, the water molecule, and the atoms that directly interact with GSH and may have an important role in the proton transfer (64-78 atoms). Density functional theory (DFT) using the B3LYP functional73,74 and the basis set 6-31G(d) was used in geometry optimization as implemented in Gaussian03.75 The low layer includes the rest of the model atoms that complete the G-site pocket. This layer was treated with the semiempirical method PM3MM.76,77 Hydrogen atoms were used as link atoms at the truncated bonds. Energy Calculations. For each enzyme a scan of the water proton approach to the GSH glutamate R-carboxylate oxygen was performed. Approximate structures of the three stationary points (reagents, transition state, and product) were taken from the scan. The stationary points were later freely optimized and

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Figure 4. Transition state for the water-assisted proton transfer: (1) wild type, (2) R15A mutant, (3) R15Rneutral mutant, and (4) R15Rε,η-cmutant. Relevant distances (nm) are shown.

their nature was confirmed by frequency calculations. After obtaining the three stationary points, we calculated the energy of the entire model with a higher theoretical level. We performed single-point calculations using DFT with the functional B3LYP for the entire system and the 6-311++G(2d,2p) basis set as implemented in Gaussian 03. A continuum model was used as an approximation to the effect of the whole protein environment as described in previous studies.53,78-80 We chose the C-PCM model81,82with a dielectric constant of 4, which is normally in conformity with the experimental protein data. Zero-point corrections and thermal and entropic effects were also added to obtain the final energies values (T ) 310.15 K, P ) 1 bar). Results and Discussion Figure 2 summarizes all the results obtained, the Gibbs energy profile of the GSH structural rearrangement plus the proton transfer Gibbs energy of the stationary points for GSTA1-1 wildtype and all mutant enzymes studied. The PMF obtained (Figure 2A) shows a minimal hysteresis (0.02 kcal · mol-1 when distance δ between atom A and B is highest and 0 kcal · mol-1 when the distance is lowest) rejecting the possibility of systematic error and emphasizing the accuracy of the calculations. The PMF calculation demonstrates that the bent GSH, with a water molecule bridging both active groups, is actually more stable than the normal opened GSH conformation. In fact, this conformational rearrangement takes place with a ∆G of -1.62 kcal · mol-1. The crystallographic structure we

used shows a water molecule hydrogen bonded to the GSH glutamyl R-carboxylate group but not bridging the two GSH active groups. So, as expected, GSH is in the open conformation. It is possible that a small barrier might exist to move the water molecule to the bridging position. The PES surface for these rearrangements is usually very flat and with multiple minima, and is surely not rate-limiting. Given the energetic proximity between the two conformations any small difference between the simulated and experimental systems can lead to a shifting toward one or the other side of the equilibrium. Figure 2B shows the DFT-calculated Gibbs activation energy, ∆G, associated with the proton transfer. The mutant enzymes show a considerable increase in the energy barrier necessary for GSH activation when comparing with the wild type, as expected. The reasons for the higher barriers are described subsequently. If we analyze all the reagents, some important remarks can be made (Figure 3). In the wild-type enzyme, the main interactions made by GSH with the active center are the following: the GSH thiol establishes a hydrogen bond with the Tyr9 side chain (Figure 3-1a); the Arg15 is bound to the GSH cysteine mainchain by a strong hydrogen bond between it Nε and the GSH carbonyl (Figure 3-1b), as well as through a strong ion-dipole interaction between it Nη and the same GSH carbonyl (Figure 3-1c); the GSH glutamyl R-carboxylate is hydrogen bonded to the Thr68 side chain (Figure 3-1d); the

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Figure 5. Products for the water-assisted proton transfer: (1) wild type, (2) R15A mutant, (3) R15Rneutral mutant, and (4) R15Rε,η-c mutant. Relevant distances (nm) are shown.

GSH glutamyl R-amino group is hydrogen bonded to the Gln67 side chain (Figure 3-1e); the GSH glycine amino group is hydrogen bonded to the subunit b Arg131 side chain (Figure 3-1f, 3-1g). In all the mutant enzymes, the substrate lost the strong interactions that were established with the Arg15 from the wild type, leading it into a different conformation. The hydrogen bonds established between the Gln67 side chain and the GSH glutamyl R-amino group in the wild-type enzyme were replaced by interactions of Gln67 with the GSH glutamyl R-carboxylate in the mutants (Figure 3-2d, 3-3d, 3-4d). Furthermore, contrarily to what happens in the wild-type enzyme, in the mutants the GSH glutamyl R-carboxylate is also hydrogen bonded to the Thr68 side chain (Figure 3-2c, 3-3c 3-4c). Figure 4 shows the transition-state structures for the enzymes analyzed. For the wild-type enzyme transition state (Figure 4-1), the interactions made by GSH within the active center are similar to the ones observed in the reagents structure (Figure 3-1). In the mutants R15A and R15Rε,η-c transition state, the Gln67 side chain is hydrogen bonded to both the carboxylate and amino groups of GSH (Figure 4-2e,f; 4-4e,f), as opposed to what happens in the respective reagents (Figure 3-2, 3-3). On the other hand, in the R15Rneutral mutant there are no changes and so the Gln67 side chain is only interacting with the GSH glutamyl R-carboxylate (Figures 4-4e). In the three mutants, the hydrogen bond observed in the reagents between the Thr68 main chain and the GSH glutamyl R-carboxylate group (Figures 3-2c, 3-3c, 3-4c) is lost in the transition state. If we compare the transition-state structure of wild-type enzyme with the mutant enzymes, we can observe that only in the first a H3O+ intermediary structure is formed (Figure 4-1b,c). In the mutant

enzymes, the water proton has to be closer to the GSH glutamate R-carboxylate oxygen (Figure 4-2σ, 4-3σ, 4-4σ) in order to promote the proton transfer, which consequently implies a higher energy barrier. In all enzymes, a shortening of the hydrogen bond between Tyr9 and the nascent thiolate is observed (Figure 4-1a, 4-2a, 4-3a, 4-4a). Analyzing the products of all the enzymes studied, the following remarks can be made (Figure 5). In the wild-type enzyme we can observe that the Thr68 side chain, which was hydrogen bonded to the GSH glutamate carboxylate oxygen, is now hydrogen bonded to the water molecule instead (Figure 5-1d). In the mutant enzymes, the initial hydrogen bond between the Thr68 side chain and the GSH glutamate carboxylate oxygen is maintained (Figure 5-2b, 5-3b, 5-4b), most likely due to different conformation of GSH. In the mutants R15A and R15Rε,η-c product structures, the Gln67 side chain is now hydrogen bonded to the glutamyl R-amino group (Figure 5-2c, 5-4c) as it happens in the three stationary states of the wildtype enzyme (Figures 3-1e, 4-1 g, 5-1e), contributing to a higher stabilization of the products. In the R15Rneutral mutant, the Gln67 side chain is only interacting with the glutamyl R-carboxylate group (Figure 5-3c). In all enzymes the amino acid Tyr9 is even closer to the sulfur atom, establishing a stronger ionic hydrogen bond interaction with it (Figure 5-1a, 5-2a, 5-3a, 5-4a). Tyr9 assumes the role of stabilizing the negative charge in the thiol sulfur atom (partial charges: wild-type S ) -0.778; R15A S ) -0.790; R15Rneutral S ) -0.794; R15Rε,η-c S ) -0.783).

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Conclusion The GSH conformational rearrangement that allows for a water molecule to interact directly with the thiol and the glutamyl R-carboxylate groups has a ∆G of -1.62 kcal · mol-1. The water-assisted proton transfers for the wild-type and the R15A mutant enzymes involve energy barriers of 13.39 and 16.77 kcal · mol-1, respectively. These values are in agreement with the experimental values for GSH-CDNB conjugation catalyzed by these enzymes (wild type ∆Gexp‡ ) 15.06 kcal · mol-1;9 R15A ∆Gexp‡ ) 17.83 kcal · mol-1 21). It should also be noted that the values calculated here are consistent with the fact that the product release is rate-limiting. For the mutants R15Rneutral and R15Rε,η-c, the energy barriers are 17.63 and 21.76 kcal · mol-1, respectively. All these results further validate the GSH activation mechanism proposed previously,53 as well as the methodology employed. The structural analyses of the enzymes allows us to conclude that in the wild-type enzyme GSH binds to the G-site pocket in a specific arrangement not seen in the mutants. The charged Arg15 establishes a strong ion-dipole interaction and a hydrogen bond with the GSH cysteine mainchain, which dictates the arrangement of the substrate. In the case of the R15Rneutral mutant, hydrogen bond interactions are still possible to be established between the residue 15 side-chain ε,η-nitrogen atoms and the GSH cysteine main-chain carbonyl group. But, as it occurred with the mutants R15A and R15Rε,η-c, eliminating the positive charge changed the orientation of residue 15 leading to a new, not catalytically efficient, GSH conformation. The experimental data available for the mutant R15A support this different GSH arrangement observed for all the mutants, as KM GSH is 10-fold increased relatively to wild-type enzyme.21 The volume of Arg15 does not seem to be as catalytically relevant as the charge. The R15Rneutral mutant, which has the same volume as the wild-type enzyme, shows a similar energy barrier with the smaller R15A mutant. For the bulkier R15Rε,η-c mutant, as expected, the energy barriers necessary to activate GSH are higher than for the other mutants. Acknowledgment. We thank the FCT (Fundac¸a˜o para a Cieˆncia e Tecnologia) for financial support linked to Project POCI/QUI/61563/2004 as well as for a Ph.D. grant SFRH/BD/ 32127/2006 for D.F.A.R.D. B.M. is supported by the Swedish Research Council. References and Notes (1) Dourado, D. F. A. R.; Fernandes, P. A.; Mannervik, B.; Ramos, M. J. Curr. Protein Peptide Sci. 2008, 9, 325. (2) 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. J. Mol. Biol. 1993, 232, 192. (3) Mannervik, B.; Jensson, H. J. Biol. Chem. 1982, 257, 9909. (4) Dirr, H.; Reinemer, P.; Huber, R. Eur. J. Biochem. 1994, 220, 645. (5) Koonin, E. V.; Mushegian, A. R.; Tatusov, R. L.; Altschul, S. F.; Bryant, S. H.; Bork, P.; Valencia, A. Protein Sci. 1994, 3, 2045. (6) Gustafsson, A.; Etahadieh, M.; Jemth, P.; Mannervik, B. Biochemistry 1999, 38, 16268. (7) Dirr, H. W.; Wallace, L. A. Biochemistry 1999, 38, 15631. (8) Allardyce, C. S.; McDonagh, P. D.; Lian, L. Y.; Wolf, C. R.; Roberts, G. C. Biochem. J. 1999, 343 (Pt 3), 525. (9) Widersten, M.; Bjo¨rnestedt, R.; Mannervik, B. Biochemistry 1996, 35, 7731. (10) Nieslanik, B. S.; Dabrowski, M. J.; Lyon, R. P.; Atkins, W. M. Biochemistry 1999, 38, 6971. (11) Lian, L. Y. Cell. Mol. Life Sci. 1998, 54, 359. (12) Ricci, G.; Caccuri, A. M.; Lo Bello, M.; Parker, M. W.; Nuccetelli, M.; Turella, P.; Stella, L.; Di Iorio, E. E.; Federici, G. Biochem. J. 2003, 376, 71.

Dourado et al. (13) Caccuri, A. M.; Antonini, G.; Board, P. G.; Parker, M. W.; Nicotra, M.; Lo Bello, M.; Federici, G.; Ricci, G. Biochem. J. 1999, 344 (Pt 2), 419. (14) Oakley, A. J.; Lo Bello, M.; Battistoni, A.; Ricci, G.; Rossjohn, J.; Villar, H. O.; Parker, M. W. J. Mol. Biol. 1997, 274, 84. (15) Cameron, A. D.; Sinning, I.; L’Hermite, G.; Olin, B.; Board, P. G.; Mannervik, B.; Jones, T. A. Structure 1995, 3, 717. (16) Raghunathan, S.; Chandross, R. J.; Kretsinger, R. H.; Allison, T. J.; Penington, C. J.; Rule, G. S. J. Mol. Biol. 1994, 238, 815. (17) Liu, S.; Zhang, P.; Ji, X.; Johnson, W. W.; Gilliland, G. L.; Armstrong, R. N. J. Biol. Chem. 1992, 267, 4296. (18) Kolm, R. H.; Sroga, G. E.; Mannervik, B. Biochem. J. 1992, 285 (Pt 2), 537. (19) Stenberg, G.; Board, P. G.; Mannervik, B. FEBS Lett. 1991, 293, 153. (20) Karshikoff, A.; Reinemer, P.; Huber, R.; Ladenstein, R. Eur. J. Biochem. 1993, 215, 663. (21) Bjo¨rnestedt, R.; Stenberg, G.; Widersten, M.; Board, P. G.; Sinning, I.; Jones, T. A.; Mannervik, B. J. Mol. Biol. 1995, 247, 765. (22) Adang, A. E.; Brussee, J.; van der Gen, A.; Mulder, G. J. Biochem. J. 1990, 269, 47. (23) Gustafsson, A.; Pettersson, P. L.; Grehn, L.; Jemth, P.; Mannervik, B. Biochemistry 2001, 40, 15835. (24) Grahn, E.; Novotny, M.; Jakobsson, E.; Gustafsson, A.; Grehn, L.; Olin, B.; Madsen, D.; Wahlberg, M.; Mannervik, B.; Kleywegt, G. J. Acta Crystallogr. DsBiol. Crystallogr. 2006, 62, 197. (25) Caccuri, A. M.; Lo Bello, M.; Nuccetelli, M.; Nicotra, M.; Rossi, P.; Antonini, G.; Federici, G.; Ricci, G. Biochemistry 1998, 37, 3028. (26) Parraga, A.; Garcia-Saez, I.; Walsh, S. B.; Mantle, T. J.; Coll, M. Biochem. J. 1998, 333 (Pt 3), 811. (27) Hubatsch, I.; Ridderstro¨m, M.; Mannervik, B. Biochem. J. 1998, 330 (Pt 1), 175. (28) Paumi, C. M.; Smitherman, P. K.; Townsend, A. J.; Morrow, C. S. Biochemistry 2004, 43, 2345. (29) Adler, V.; Yin, Z.; Fuchs, S. Y.; Benezra, M.; Rosario, L.; Tew, K. D.; Pincus, M. R.; Sardana, M.; Henderson, C. J.; Wolf, C. R.; Davis, R. J.; Ronai, Z. EMBO J. 1999, 18, 1321. (30) Johansson, A.-S.; Mannervik, B. J. Biol. Chem. 2001, 276, 33061. (31) Sugioka, Y.; Fujii-Kuriyama, Y.; Kitagawa, T.; Muramatsu, M. Cancer Res. 1985, 45, 365. (32) Satoh, K.; Kitahara, A.; Soma, Y.; Inaba, Y.; Hatayama, I.; Sato, K. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3964. (33) Ikeda, H.; Nishi, S.; Sakai, M. Biochem. J. 2004, 380, 515. (34) Okuda, A.; Imagawa, M.; Sakai, M.; Muramatsu, M. EMBO J. 1990, 9, 1131. (35) Green, J. A.; Vistica, D. T.; Young, R. C.; Hamilton, T. C.; Rogan, A. M.; Ozols, R. F. Cancer Res. 1984, 44, 5427. (36) Gupta, V.; Jani, J. P.; Jacobs, S.; Levitt, M.; Fields, L.; Awasthi, S.; Xu, B. H.; Sreevardhan, M.; Awasthi, Y. C.; Singh, S. V. Cancer Chemother. Pharmacol. 1995, 36, 13. (37) Hamilton, T. C.; Winker, M. A.; Louie, K. G.; Batist, G.; Behrens, B. C.; Tsuruo, T.; Grotzinger, K. R.; McKoy, W. M.; Young, R. C.; Ozols, R. F. Biochem. Pharmacol. 1985, 34, 2583. (38) Hall, A.; Robson, C. N.; Hickson, I. D.; Harris, A. L.; Proctor, S. J.; Cattan, A. R. Cancer Res. 1989, 49, 6265. (39) Tsuchida, S.; Sato, K. Crit. ReV. Biochem. Mol. Biol. 1992, 27, 337. (40) Tew, K. D.; Monks, A.; Barone, L.; Rosser, D.; Akerman, G.; Montali, J. A.; Wheatley, J. B.; Schmidt, D. E., Jr. Mol. Pharmacol. 1996, 50, 149. (41) Waxman, D. J. Cancer Res. 1990, 50, 6449. (42) Niitsu, Y.; Takahashi, Y.; Ban, N.; Takayama, T.; Saito, T.; Katahira, T.; Umetsu, Y.; Nakajima, T.; Ohi, M.; Kuga, T.; Sakamaki, S.; Matsunaga, T.; Hirayama, Y.; Kuroda, H.; Homma, H.; Kato, J.; Kogawa, K. Chem. Biol. Interact. 1998, 111-112, 325. (43) Lewis, A. D.; Hayes, J. D.; Wolf, C. R. Carcinogenesis 1988, 9, 1283. (44) Kuzmich, S.; Vanderveer, L. A.; Walsh, E. S.; LaCreta, F. P.; Tew, K. D. Biochem. J. 1992, 281 (Pt 1), 219. (45) Mannervik, B.; Awasthi, Y. C.; Board, P. G.; Hayes, J. D.; Di Ilio, C.; Ketterer, B.; Listowsky, I.; Morgenstern, R.; Muramatsu, M.; Pearson, W. R. Biochem. J. 1992, 282 (Pt 1), 305. (46) Ouwerkerk-Mahadevan, S.; Mulder, G. J. Chem. Biol. Interact. 1998, 111-112, 163. (47) Cacciatore, I.; Caccuri, A. M.; Di Stefano, A.; Luisi, G.; Nalli, M.; Pinnen, F.; Ricci, G.; Sozio, P. Farmaco 2003, 58, 787. (48) Burg, D.; Filippov, D. V.; Hermanns, R.; van der Marel, G. A.; van Boom, J. H.; Mulder, G. J. Bioorg. Med. Chem. 2002, 10, 195. (49) Dourado, D. F. A. R.; Fernandes, P. A.; Mannervik, B.; Ramos, M. J. Theor. Chem. Acc. 2009, 124, 71. (50) Nieslanik, B. S.; Atkins, W. M. J. Am. Chem. Soc. 1998, 120, 6651. (51) Adang, A. E.; Brussee, J.; Meyer, D. J.; Coles, B.; Ketterer, B.; van der Gen, A.; Mulder, G. J. Biochem. J. 1988, 255, 721.

Glutathione Transferase A1-1 (52) Adang, A. E.; Meyer, D. J.; Brussee, J.; Van der Gen, A.; Ketterer, B.; Mulder, G. J. Biochem. J. 1989, 264, 759. (53) Dourado, D. F. A. R.; Fernandes, P. A.; Mannervik, B.; Ramos, M. J. Chem.sEur. J. 2008, 14, 9591. (54) Berman, H. M.; Battistuz, T.; Bhat, T. N.; Bluhm, W. F.; Bourne, P. E.; Burkhardt, K.; Feng, Z.; Gilliland, G. L.; Iype, L.; Jain, S.; Fagan, P.; Marvin, J.; Padilla, D.; Ravichandran, V.; Schneider, B.; Thanki, N.; Weissig, H.; Westbrook, J. D.; Zardecki, C. Acta Crystallogr. DsBiol. Crystallogr. 2002, 58, 899. (55) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179. (56) Wang, J. M.; Cieplak, P.; Kollman, P. A. J. Comput. Chem. 2000, 21, 1049. (57) Lindahl, E.; Hess, B.; Van der Spoel, D. J. Mol. Mod. 2001, 7, 306. (58) Sorin, E. J.; Pande, V. S. Biophys. J. 2005, 88, 2472. (59) Berendsen, H., Postma, J. P. M. van Gunsteren, W. F. Intermolecular Forces; D. Reidel Publishing Co.: Dordrecht, The Netherlands, 1981. (60) Berendsen, H.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3584. (61) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577. (62) Torrie, G. M.; Valleau, J. P. J. Comput. Phys. 1977, 23, 187. (63) Kumar, S.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A.; Rosenberg, J. M. J. Comput. Chem. 1992, 13, 1011. (64) Bakowies, D.; Thiel, W. J. Phys. Chem. 1996, 100, 10580. (65) Dapprich, S.; Komaromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J. J. Mol. Struct. Theochem 1999, 462, 1.

J. Phys. Chem. B, Vol. 114, No. 4, 2010 1697 (66) Cosman, P. C.; Rogers, J. K.; Sherwood, P. G.; Zeger, K. IEEE Trans. Image Process 2000, 9, 982. (67) Sousa, S. F.; Fernandes, P. A.; Ramos, M. J. Proteins Struct., Funct., Bioinf. 2007, 66, 205. (68) Sousa, S. F.; Fernandes, P. A.; Ramos, M. J. J. Comput. Chem. 2007, 28, 1160. (69) Cerqueira, N. M. F. S. A.; Fernandes, P. A.; Eriksson, L. A.; Ramos, M. J. Biophys. J. 2006, 90, 2109. (70) Cerqueira, N. M. F. S. A.; Fernandes, P. A.; Eriksson, L. A.; Ramos, M. J. J. Comput. Chem. 2004, 25, 2031. (71) Carvalho, A. T.; Fernandes, P. A.; Ramos, M. J. J. Phys. Chem. B 2006, 110, 5758. (72) Carvalho, A. T.; Fernandes, P. A.; Ramos, M. J. J. Comput. Chem. 2006, 27, 966. (73) Lee, C. T.; Yang, W. T.; Parr, R. G. Phys. ReV. B Condens. Matter 1988, 37, 785. (74) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (75) Frisch, M. J. Gaussian 03; Gaussian Inc.: Wallingford, CT, 2004. (76) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 209. (77) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 221. (78) Siegbahn, P. E. M. Abstr. Pap. Am. Chem. Soc. 1998, 216, U101. (79) Siegbahn, P. E. M. J. Am. Chem. Soc. 1998, 120, 8417. (80) Blomberg, M. R. A.; Siegbahn, P. E. M.; Babcock, G. T. J. Am. Chem. Soc. 1998, 120, 8812. (81) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995. (82) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V. J. Comput. Chem. 2003, 24, 669.

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