Hydrolysis of the E2P Phosphoenzyme of the Ca2+-ATPase: A

Jun 26, 2013 - Dephosphorylation of the E2P phosphoenzyme intermediate of the sarcoplasmic reticulum Ca2+-ATPase was studied using density functional ...
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Hydrolysis of the E2P Phosphoenzyme of the Ca2+-ATPase: A Theoretical Study Maria E. Rudbeck,*,† Margareta R. A. Blomberg,‡ and Andreas Barth*,† †

Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, 10691 Stockholm, Sweden Department of Organic Chemistry, Arrhenius Laboratories, Stockholm University, 10691 Stockholm, Sweden

‡

S Supporting Information *

ABSTRACT: Dephosphorylation of the E2P phosphoenzyme intermediate of the sarcoplasmic reticulum Ca2+-ATPase was studied using density functional theory. The hydrolysis reaction proceeds via a nucleophilic attack on the phosphorylated residue Asp351 by a water molecule, which is positioned by the nearby residue Glu183 acting as a base. The activation barrier was calculated to be 14.3 kcal/mol, which agrees well with values of 15−17 kcal/mol derived from experimentally observed rates. The optimized structure of the transition state reveals considerable bond breakage between phosphorus and the Asp351 oxygen (distance 2.19 Å) and little bond formation to the attacking water oxygen (distance 2.26 Å). Upon formation of the singly protonated phosphate product, Glu183 becomes protonated. The bridging aspartyl phosphate oxygen approaches Lys684 progressively when proceeding from the reactant state (distance 1.94 Å) via the transition state (1.78 Å) to the product state (1.58 Å). This stabilizes the negative charge that develops on the leaving group. The reaction was calculated to be slightly endergonic (+0.9 kcal/mol) and therefore reversible, in line with experimental findings. It is catalyzed by a preorganized active site with little movement of the nonreacting groups except for a rotation of Thr625 toward the phosphate group.

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INTRODUCTION The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)1 is the best studied P-type ATPase. At the expense of ATP, the enzyme transports two Ca2+ ions from the cytoplasm into the sarcoplasmic reticulum lumen. Phosphorylation at Asp351 leads to two phosphoenzyme intermediates: the ADP-sensitive Ca2E1P and the ADP-insensitive E2P. The Ca2+ ions are released into the lumen during the conversion between these intermediates (Ca2E1P to E2P), and two to three H+ are bound instead. Subsequently, E2P is hydrolyzed to E2. All steps in the catalytic cycle are reversible, implying that E2 can be phosphorylated by inorganic phosphate Pi to give E2P.2,3 One of the intriguing properties of E2P is its rapid hydrolysis, which contrasts with the slow hydrolysis of the model compound acetyl phosphate in aqueous solution. The hydrolysis rates of E2P vary depending on the experimental conditions (pH, temperature, and ion concentrations) and are between 1 and 115 s−1 in the temperature range of 20−30 °C.4−12 Using transition state theory, these rates can be translated into Gibbs free energies and correspond to activation barriers of 15−17 kcal/mol. The fast enzymatic catalysis of the Ca2+-ATPase dephosphorylation is an essential feature of the enzyme, which ensures fast muscle relaxation. A subject much discussed is whether phosphoric monoesters undergo phosphate transfer via associative or dissociative mechanisms.13−18 Most reaction mechanisms are neither fully associative nor fully dissociative and proceed via a concerted mechanism without an intermediate. The transition state (TS) © 2013 American Chemical Society

in the mechanism can then have varying degrees of dissociative character, depending on the scissile P−O bond strength. Phosphate monoester dianions in solution generally react via a TS that is largely dissociative in character, i.e., with considerable bond breakage of the scissile P−O bond and only little bond formation to the attacking nucleophile. The same seems to be true for many enzymatic reactions.19 However, for a number of enzymes, a more associative character of the TS has been suggested15 and the mechanism of enzymatic phosphate reactions continues to be discussed. So far, the mechanism of aspartyl phosphate hydrolysis of two enzymes has been studied computationally. Both belong to the haloacid dehalogenase (HAD) superfamily as does the phosphorylation domain of SERCA. Re et al. studied the nature of the TS of phosphoserine phosphatase (PSP) using hybrid quantum mechanics/molecular mechanics (QM/MM).20 Their calculations suggest that the TS has a geometrically associative but a electronically dissociative character. Himo et al.21 studied the reaction mechanism of human deoxyribonucleotidase (dN) using a QM model with 99 atoms. Their study tried to locate a pentacoordinated intermediate, which had earlier been suggested,22 with no success.21 The active sites of all HAD proteins are very similar; still, the energetics of the hydrolysis reaction obtained by Re et al20 is Received: May 21, 2013 Revised: June 20, 2013 Published: June 26, 2013 9224

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Figure 1. The reactant with (model1) and without Gly626 (model2). R1−R3 are the bond lengths in angstroms. Atoms marked by red circles represent backbone atoms with fixed positions.

atoms are usually fixed at the positions of the experimental structures. This maintains the backbone structure and prevents unnatural movements, keeping the optimized structure close to the experimental structure.29,31 However, it also prevents a possible movement of these atoms during the reaction and might make the model too rigid. Again, a larger model size allows for more internal flexibility within the cluster and makes the restrictions less significant.29 The active site of E2P was constructed from the crystal structure of E2·Pi (PDB: 1WPG24) with the MgF42‑ analog; this structure was chosen since it has the best resolution (2.30 Å) and the TGES-loop is pointing toward the active site. The phosphate analogue MgF42− was substituted for phosphate and a water molecule. The residues and water molecules that were expected to be of importance were extracted from the PDB-file together with the coordinating metal ion. Our main model (model1; see Figure 1) includes Asp351 bound to the phosphate, a magnesium ion, five water molecules (two of which were ligated to Mg2+), and the following residues: Thr181, Gly182, Glu183, Thr353, Thr625, Gly626, Lys684, Asp703, Asn706, and Asp707. To further reduce the model size, the residues were truncated so that only the side chains and those backbone atoms that interact with other atoms of the model were kept. For the peptide groups that were not considered to be important, the peptides carbonyl carbon and nitrogen were substituted for hydrogen atoms. These hydrogens and the α-carbon atoms were fixed to the corresponding X-ray coordinates (marked by red circles in Figure 1). The side chains of glutamate, aspartate, and lysine were all charged, which is in accordance with the physiological pH at 7.4. The total charge of the system was −2, which is the charge of the phosphate substrate and is in accordance with the model used by Himo et al21 studying a similar system, and the state is a closed shell singlet. Our main model, termed model1, contained Gly626, since mutation Gly626Ala severely affects the formation of E2P from inorganic phosphate, which was explained by steric collisions with Thr181.32 Initially, calculations were also performed on a model without Gly626, termed model2, which contained two

very different from that obtained by Himo et al.21 The calculated activation barrier of PSP is high and the reaction is found to be very endothermic,20 while the activation barrier of dN is of reasonable height and the reaction is less endothermic.21 In the present study, we investigated the mechanism for the hydrolysis of E2P employing QM models larger than any of the previous studies (∼150 atoms). An important methodological aspect of the present study is also that dispersion effects were included, using the empirical formula by Grimme.23 The models used here were based on the crystal structure of an E2·Pi analogue, which represents the product state just after the hydrolysis of the aspartyl phosphate but before the release of phosphate from the ATPase.24 There exist several crystal structures of E2P-like states, which are all based on phosphate analogues: BeF3−, AlF4− or MgF42‑.24−27 The BeF3− analogue models the E2P ground state, the AlF4− analogue models the TS, and the MgF42‑ analogue models the product state. The crystal structure of the ground state is in an inactive mode,28 where Gly182 blocks the access of an attacking water molecule to the phosphate analogue. In order to activate this structure for the dephosphorylation reaction, the 181TGES signature sequence needs to be reoriented, which positions a water molecule in an attacking mode.25,27 The reaction considered here starts after the rearrangement of the TGES-loop.

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METHODS Active Site Models. The active site was modeled using the quantum chemical cluster approach.29 The idea is to perform accurate QM calculations on a small but carefully selected fragment of the protein and calculate an energy profile for the protein reaction. This approach describes the local effects in the catalytic site at a high level of theory, but does not include longrange electrostatic interactions with protein parts outside the cluster. However, for large models, like the ∼150 atom models used here, the calculated energies agree with experimental data and become independent of solvation effects, indicating that the cluster size has converged.29,30 In order to approximate the steric constraints of the surroundings, some of the peripheral 9225

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Table 1. Enthalpy Changes for the Dephosphorylation of E2P Model1a reactant TS product a product b

ΔHsdz kcal/molb

ΔH kcal/molc

ΔΔHs kcal/mold

ΔΔHd kcal/mole

ΔΔHz kcal/molf

ΔΔHsdz kcal/molg

0.0 14.3 0.9 −9.5

0.0 21.4 6.9 −3.2

0.0 −1.7 0.8 −0.6

0.0 −5.0 −6.3 −4.5

0.0 −0.4 −0.5 −1.2

0.0 −7.1 −6.0 −6.3

Because changes in entropy are expected to be small (see Methods), the calculated ΔH values are interpreted as free energy changes ΔG. bChange in enthalpy relative to the reactant obtained in calculations that included dispersion forces, solvation (ε = 4) and zero-point energies. cChange in enthalpy calculated without dispersion forces, solvation and zero-point energies. dContribution of solvation (ε = 4) to the change in enthalpy. e Contribution of dispersion forces to the change in enthalpy. fContribution of zero-point energies to the change in enthalpy. gΔΔHs + ΔΔHd + ΔΔHz a

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RESULTS AND DISCUSSION Reactant. Our model is based on the crystal structure, which represents the E2·Pi state, adopted just after the hydrolysis of the aspartyl phosphate but before the release of phosphate from the ATPase. The first aim was therefore to optimize the reactant for the hydrolysis reaction. The optimized geometry of the reactant is shown in Figure 1, where the phosphate group is bound to Asp351 (R1=1.82 Å). Relevant distances of this model are given in Table 2. The distance

water molecules instead of Gly626. Selected results obtained with model2, related to a possible role for Gly626, are also presented. Computational Details. All geometries and energies presented were calculated using the B3LYP density functional theory method as implemented in the Jaguar 7.6 program.33 Geometry optimizations were performed using the 6-31G** basis set, and single-point calculations with the larger basis set cc-pVTZ(-f) (6-311G* for Mg2+) were done to obtain more accurate energies. Solvation energies were added as single-point calculations using the Poisson−Boltzmann solvation model implemented in Jaguar. In this model a cavity around the system is surrounded by a polarizable dielectric continuum with a dielectric constant here set to 4, modeling a protein environment.31 The solvent effects on the relative energies are listed in Table 1. Solvent effects lowered the activation barrier by 1.7 kcal/mol for model1. Both dispersion and zero-point corrections were included and are listed in Table 1. The dispersion corrections were calculated with the B3LYP-optimized structures according to the DFT-D2 method23 implemented in the XYZ-viewer.34 The calculated energies are a good approximation of those obtained when the geometry is optimized under consideration of dispersion forces.35 The zero-point energies were obtained from the Hessians, i.e., the second derivatives with respect to nuclear motion, which were calculated using the Gaussian 09 package.36 The dispersion and zero-point energy corrections for the TS and product structures were found to be larger than those for the reactants. For model1, this resulted in a 5.4 kcal/ mol lower activation barrier and a lowering of the energy of the product relative to that of the reactant by 6.8 kcal/mol. Of the three contributions from solvation, zero-point energy, and dispersion, the latter had the largest influence on the energy profile (Table 1). Approximate TSs were obtained by optimizing different constrained distances of the O−P bonds to be formed or cleaved (and O−H for the second TS in the second model). Full TS-optimizations were then performed starting from the structure with the highest energy using Gaussian 09. The TS vibrational frequencies were then analyzed to ensure that there was only one imaginary frequency corresponding to the correct character of the TS and that the remaining imaginary frequencies were due to the fixed coordinates. Since several atoms were locked into certain coordinates, an accurate entropy could not be calculated. The ΔG values were therefore approximated with the enthalpy. However, the effects of the entropy are considered to be small since there are no molecules entering or leaving during the reaction steps studied.

Table 2. Relevant Distances for Model1 Distance/Å bond or interaction Phosphate Bonds R1: P−O1 R2: P−attacking water oxygen (O2) P−OT127 P−OT129 P−OT130 Average P−OT Interactions with Phosphate Thr181 (CO)−phosphate (O2H) Glu183 (OH)−O2 Thr625 (OH)−OT129 Lys684 (NH)−O1 Lys684 (NH)−OT127 Asn706 (NH)−OT127 water (OH)−OT127 water coordinating to Mg2+ (OH)−OT127 attacking water (O2H)−OT129 Mg2+−OT130 Other Interactions Thr181 (CO)−attacking water (O2H) R3: Glu183 (O3)−attacking water (O2H) Asp351 (CO)−Mg2+ Thr353 (OH)−Glu183 (O3) Thr625 (OH)−attacking water (O2)

reactant

TS

product1a

1.82 4.08 1.544 1.506 1.528 1.526

2.19 2.26 1.541 1.501 1.522 1.521

3.00 1.72 1.564 1.520 1.534 1.539

3.42 1.94 2.75 2.25 1.74 1.82 1.85 2.04

1.74 1.78 2.93 2.44 1.60 1.92 2.08

1.68 1.52 1.67 1.58 3.26 2.39 1.63 1.80 2.09

3.82 1.73 2.14 1.71 1.74

1.77 1.67 2.08 1.70 2.76

1.03 2.02 1.90 3.50

The terminal phosphate oxygens are numbered as in the structural files provided as Supporting Information. OT127 corresponds to the lower left phosphate oxygen in Figure 1, OT129 is the phosphate oxygen that points to the right in the figure, and OT130 coordinates to Mg2+.

between the nucleophile, an attacking water molecule that we will refer to as water1, and the phosphorus of the phosphate group is 4.08 Å (R2). Water1 is kept in place by hydrogen bonds to Glu183 (R3 = 1.73 Å), to Thr625 (H···O 1.74 Å) and to one of the terminal phosphate oxygens (H···O 1.85 Å). Transition State. The TS of hydrolysis was localized, its optimized structure is shown in Figure 2, and relevant distances 9226

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bond breakage between phosphorus and the Asp351 oxygen and only little bond formation to the attacking water oxygen. Judged from the average terminal P−O bond length, which is shorter in the TS than in the reactant state (1.521 Å versus 1.526 Å), the interactions with the terminal phosphate oxygens are slightly weaker in the TS than in the reactant state. Shorter bonds and weaker interactions in the TS are expected when the negative charge on the terminal oxygens is reduced in a more metaphosphate-like (PO3−) structure. We note however, that this effect is very small. Apart from movements of the reacting molecules, most of the active site is rather rigid. An exception is the side chain of Thr625, which follows the movement of the attacking water molecule and rotates toward the phosphate group to form a hydrogen bond with one of the terminal phosphate oxygens (H···O 1.74 Å). Product. Figure 3 shows the optimized product where water has lost one proton to Glu183 (R3 = 1.03 Å) and covalently binds to the phosphate group (R2 = 1.72) giving rise to a singly protonated phosphate. This product will be referred to as product1a from now on. The distance between the Asp351 oxygen O1 and the phosphorus atom is 3.00 Å (R1). Apart from the movement of the phosphate group, there is very little movement in the catalytic site, and the interactions are similar to those in the TS. Exceptions are Lys684, which interacts stronger with the leaving oxygen of Asp351 and weaker with a terminal phosphate oxygen in product1a, and Thr353, which interacts weaker with Glu183 (see Table 2). Energy Profile. The energy profile for the hydrolysis reaction is shown in Figure 4, and relative enthalpy values are listed in Table 1. They are interpreted as free energy values, since entropy contributions are expected to be small (see Methods). The overall reaction is calculated to be almost thermoneutral (+0.9 kcal/mol), indicating that the reaction is reversible, which is in agreement with the reaction cycle of Ca2+-ATPase where all steps are reversible. Experimentally, E2P dephosphorylation (E2P → E2·Pi) was found to be close to

Figure 2. The optimized transition-state of model1. R1−R3 are the bond lengths in angstroms as defined in Figure 1.

are given in Table 2. The energy barrier of this TS was calculated to be 14.3 kcal/mol, and its imaginary wavenumber was found at 166i cm−1. Water1 has now approached the phosphate group (R2 = 2.26 Å), and the hydrogen bond to Glu183 is stronger (R3 = 1.67 Å), whereas that to Thr625 is broken (H···O 2.76 Å). The water has rotated and moved so that the water oxygen faces the phosphorus atom for an in-line attack. In consequence, the hydrogen bond between water and the terminal phosphate oxygen in the reactant is replaced by one with the backbone carbonyl of Thr181 (H···O 1.77 Å) in the TS. The distance between Asp351 and the phosphate group has increased to 2.19 Å (R1). These distances to the leaving and the attacking oxygen atoms indicate a TS with considerable

Figure 3. Product1a (left) and product1b (right) of model1. In product1a, Glu183 is protonated, and the second proton of water1 is hydrogen bonded to Thr181. In product1b, the phosphate molecule is doubly protonated with one proton hydrogen bonding to Glu183 and the second to Asp351. 9227

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Figure 4. The calculated energy profile for the different steps in the hydrolysis of E2P.

thermoneutral (+0.3 kcal/mol).7 Experimental rates predict an activation barrier of 15−17 kcal/mol, which is somewhat higher than our calculated barrier of 14.3 kcal/mol. This is expected, since the reactant in our calculations does not correspond to the E2P ground state, which is the initial state in the experiments. The E2P ground state is presumed to differ from the reactant state by the position of the TGES loop as discussed in the Introduction. There is no evidence for a significant population of the reactant structure in the ensemble of structures that constitute the E2P state from the infrared spectrum of the E2P phosphate group (Rudbeck et al., manuscript in preparation). This indicates that the reorientation of the TGES-loop from the ground state to our reactant model costs a few kcal/mol, and therefore that the barrier between E2P and the TS is larger than the barrier between reactant and TS calculated here. The Role of Individual Groups in the Reaction Mechanism. Our calculations confirm that Glu183 acts as a base in the hydrolysis reaction as was previously concluded from structural data,24 indicating that Glu183 is protonated in the E2·MgF42− crystal, and from the effects of mutation of Glu183.9 Most mutations of Glu183 dramatically reduce the dephosphorylation rate. Only mutation to Asp enables a relatively rapid reaction.8 This demonstrates that a carboxylate group is necessary for fast dephosphorylation of E2P. The important role of this residue is also reflected in its high conservation in the ATPase protein family.37 E2P is more stable at pH 6 than at pH 7 due to an inhibition of its dephosphorylation,5,38 and this has been tentatively explained by the protonation of protein residues acting as a general base in the catalytic mechanism.38 Considering the reaction mechanism presented here, it is tempting to speculate that this effect is due to protonation of Glu183 already in the TS. Protonation will weaken the interaction with the hydrogen atom of the attacking water molecule in the TS and therefore increase its energy. Furthermore, a protonated Glu183 will not be available as a base to accept one of the water protons. If this role of Glu183 in controlling the dephosphorylation rate is correct, its pKa should be around 6, i.e., elevated with respect to carboxyl groups exposed to water (pKa ≈ 4) because of a less hydrophilic environment. This is conceivable, as discussed in the following. In the reactant state, one of the Glu183 carboxylate oxygens interacts with the attacking water and Thr353, the other is exposed to a water-filled cavity and interacts with two crystal waters (O···O distances 1.7 Å)

according to the crystal structure.24 These strong interactions indicate that Glu183 is ionized at pH 7. However, the closeness of the negatively charged phosphate group and the low dielectric constant of the surrounding protein environment make it likely that the pKa of Glu183 is somewhat higher than in an aqueous environment, in line with its proposed role in controlling the dephosphorylation rate of E2P. In addition to Glu183, Thr625 is important in binding the attacking water molecule in the reactant. Furthermore, it stabilizes the transition and product states by an interaction with a terminal phosphate oxygen as described above. These dual roles are accomplished by a rotation of the Thr625 side chain. The significance of Thr625 for the dephosphorylation reaction in our computations agrees with its conservation in the haloacid dehalogenase superfamily.39,40 and with experimental evidence for its importance for the back reaction: substitution of Thr625 by Ala abolishes phosphorylation from inorganic phosphate.32 Due to the severe effect of the substitution on phosphoenzyme formation, its effect on the dephosphorylation rate of E2P could not be assessed experimentally. The hydroxyl group of Thr353 interacts with the carboxyl oxygen of Glu183, which points toward the attacking water molecule in the reactant and the TS. The hydrogen bond stabilizes the negative charge on the Glu183 oxygen and thus enhances the interaction with the attacking water molecule, which facilitates the transfer of the water proton. In the product, Thr353 interacts with the protonated carboxyl oxygen of Glu183 (O3). The hydrogen bond with Thr353 is stronger in the reactant (H···O distance 1.71 Å, OH···O angle 172°) and transition (1.70 Å, 167°) states than in the product state (1.90 Å, 162°), indicating that Thr353 lowers the reactant and the TS energy relative to that of the product. Thr353 is conserved within the P-type ATPase family37 and its substitution abolishes phosphorylation by inorganic phosphate (the back reaction of the dephosphorylation studied here) in most cases. The exception is a replacement by Ser, which also carries a hydroxyl group, in which case the dephosphorylation of E2P is considerably accelerated.41 In both amino acids, the hydroxyl group is attached to the Cβ atom, but the Thr side chain is larger, which might prevent optimal interactions in the TS due to steric hindrance. The results of side chain substitutions indicate the important role of the hydroxyl group in line with our calculations. Another important residue for the dephosphorylation of the E2P phosphoenzyme is Lys684. The distance between its 9228

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experiments. As this cannot be modeled with our approach, we conclude that our product state represents the state immediately after the dephosphorylation reaction; it is one of several structures that constitute the E2·Pi state. In order to explain why product1b is not formed upon reorientation of the phosphate group, we speculate that the conformational change needed to enable reorientation of the phosphate group exposes Glu183 to a more polar environment, which leads to its deprotonation. Thus, the singly protonated phosphate group would reorient while Glu183 is deprotonated. Such an environmental change happens when A and P domains slightly detach, as it occurs in the transition from the E2·Pi analogue state E2·MgF42− to the subsequent E2 state.24 In E2, the carboxyl group of Glu183 faces the amino group of Gln202 and there is space for water molecules at both carboxyl oxygens for further interactions.45 Therefore, Glu183 is expected to be deprotonated in E2. Upon tight reassociation of A and P domains to return to the E2·Pi state modeled here, the phosphate is supposed to interact initially mainly with the TGES loop. These interactions are strongest when Glu183 becomes protonated and interacts with the protonated phosphate oxygen since this allows for two hydrogen bonds to be formed (to Glu183 and Thr181) as in product1a. This proposed mechanism avoids the formation of product1b, which would be a dead end complex in the back reaction to E2P due to its low energy. The Role of Restriction of Water Access. Restriction of water access to the catalytic site might be an important property of the catalytic site. In this context, we briefly discuss here model2 containing two extra water molecules instead of Gly626. In this model, the space around the phosphate group is less tight, allowing more water molecules to interact with the phosphate group. Gly626 resides in a loop that connects an αhelix and a β-strand and encloses the catalytic site from one side. Thus our model2 is a simple way to simulate a different positioning of this loop, further away from the active site and allowing water access to the phosphate group. In more general terms, the model simulates an active site, which is less shielded against the aqueous environment. The hydrolysis step of the reaction occurred in a similar way in model2 as in model1 with an energy barrier of 10.4 kcal/mol (14.3 kcal/mol for model1). The most exergonic product structure was found to be product2b, similar to product1b of model1. In contrast to model1, a reaction pathway was located for model2 that allowed transfer of the second proton and formation of product2b, made possible by the involvement of the surrounding water molecules. The energy barrier of the transfer was 11.1 kcal/mol relative to product2a, and product2b was calculated to be exergonic by 16.2 kcal/mol relative to the reactant, resulting in an energy barrier of 26.6 kcal/mol for the overall back reaction to the reactant. Since all reactions steps in the Ca2+-ATPase are reversible, this barrier is too high. We conclude from these calculations that the effect of the Gly626 backbone, included in model1 and not in model2, might be to hide the phosphate group from more interacting water molecules and thereby hinder the transport of the proton and the formation of the very exergonic product1b. This implies in more general terms that restriction of water access to the catalytic site prevents formation of a doubly protonated phosphate group and guarantees the reversibility of the pump. Comparison to Previous Calculations. Up to now, dephosporylation reactions of three members of the HAD protein family have been studied theoretically: SERCA (this

closest hydrogen and the bridging oxygen atom of Asp351 decreases from 1.94 Å in the reactant state to 1.78 Å in the TS and to 1.58 Å in the product state. Interestingly, these distance changes are not achieved by movement of Lys684 but rather by minor movements of the Asp351 carboxyl group. The stronger interaction in the course of the reaction between the positively charged Lys684 and the Asp351 oxygen stabilizes the negative charge on the leaving group that develops in the TS and is present in the product state. This important role of Lys684 is in line with our previous study of environmental effects on phosphorylated molecules,28 which has shown that an interaction with the bridging oxygen elongates the scissile P− O bond and thus is an important means to weaken this bond. Lys684 is conserved in the haloacid dehalogenase superfamily39,40 and substitutions of its side chain abolish phosphorylation by inorganic phosphate.42 Substitution for Arg allows phosphorylation of the ATPase by ATP in the forward mode of the reaction cycle and formation of E2P. In this case, dephosphorylation of E2P seems to be as rapid as for the wild type.42 This indicates that if residue 684 carries a positive charge, the dephosphorylation reaction is similar to that of the wild type. The magnesium ion coordinates to the carboxyl oxygen of Asp351 and to one of the terminal phosphate oxygens throughout the reaction with maximum distance changes of ∼0.1 Å. The largest change is found for the interaction with the Asp351 oxygen, and the distance is largest in the reactant state. The terminal phosphate oxygen which coordinates to Mg2+ is the one that moves least in the reaction. The angle between carbonyl oxygen, Mg2+, and terminal oxygen is strained in the reactant state (84°) and the product state (95°) but relaxes toward an ideal tetragonal symmetry in the TS (90°). A Doubly Protonated Phosphate Product? In the initial search for the most stable product, an alternative to product1a was found, 9.5 kcal/mol lower in energy than the reactant (product1b; see Figures 3 and 4 and Table 1). The difference between the two products is the position of water1′s two protons. In product1a, water1 has lost one of its protons to Glu183, and its second proton is hydrogen bonded to the carbonyl group of Thr181. In product1b, the phosphate group is doubly protonated: the first proton hydrogen bonds to Glu183 and the second to Asp351 (the leaving group). The lower energy of product1b leads to a reverse activation barrier of 23.8 kcal/mol, which is too high for a reversible reaction. We tried to find a reaction pathway between product1a and product1b but did not manage to locate a TS. The calculated energies for the pathways explored were very high, indicating that product1b is not reachable from product1a using this model. We note that the product state structure is likely more heterogeneous than what could be assessed in our calculations. Evidence for this comes from 18O/16O isotope exchange of the E2P phosphate oxygens.12,43,44 The complete exchange of the phosphate oxygens observed experimentally in consecutive dephosphorylation and phosphorylation cycles requires a reorientation of the phosphate group as well as an exchange of the product water by a different water molecule. Reorientation of the phosphate group is difficult to conceive within the frame of our calculations, since the terminal phosphate oxygens are held in place by up to three hydrogen bonds, which imposes a high activation barrier for rotation of the phosphate group. Thus conformational flexibility of E2·Pi seems to be required to explain the isotope exchange 9229

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work), dN21 and PSP.20 The proteins have different functions but similar active sites. The dephosphorylation step of all the proteins includes a phosphorylated aspartate as a reactant, a Mg-ion, an attacking water, and a carboxylate base (Glu183 in our system). Our optimized TS structure is symmetric regarding the distances of the phosphorus atom to the leaving Asp351 oxygen (R1 = 2.19 Å) and to the attacking water oxygen (R2 = 2.26 Å). These distances indicate a late (product-like) TS characterized by considerable bond breakage between phosphorus and the Asp351 oxygen and only little bond formation to the attacking water oxygen. Our values are intermediate between those of the previous calculations,20,21 which resulted in distances of 1.9121 and 2.52 Å20 for P−OAsp and 2.7821 and 1.92 Å20 for P−Owater. Part of the variation between the different studies may be explained by the different computational approaches. The small basis set used by Himo et al.21 during the geometry optimization resulted in reaction coordinates of an early (reactant-like) TS. From their own single-point calculations with a larger basis set, the TS is later with reaction coordinates similar to ours (2.13 Å for P−OAsp and 2.2 Å for for P− Owater).21 Due to the similarity of the active sites, similar energy profiles for the reaction are expected for the three members of the HAD protein family. However, this is not the case, and we discuss in the following methodological differences as a possible explanation. Himo et al. studied the reaction mechanism of the phosphate hydrolysis in dN using a model with 99 atoms and no constraints.21 The model was fully optimized using B3LYP/ LANL2DZ, and single-point energies were calculated using B3LYP/6-311+G(2d,2p). Solvation energies were added but no dispersion or zero-point energy corrections. The activation barrier calculated by Himo et al. is 12.5 kcal/mol, and their product is endothermic by 7.1 kcal/mol. The latter is the main discrepancy between their work and ours. However, similar dispersion effects as in our case (see Computational Details and Table 1) would make the product of Himo et al. nearly thermoneutral. The activation barrier is influenced by several computational aspects: (i) it is lowered by dispersion effects if they are similar to those found in this work, (ii) it is increased by a larger basis set,21 and (iii) solvation has less effect in larger QM models.29,46−48 Thus, we explain the differences between our calculations and those by Himo et al. by our consideration of dispersion interactions, our larger basis set, and our larger model. It is more difficult to speculate on the difference between our work and the study by Re et al.20 of the hydrolysis of phosphoL-serine that is catalyzed by PSP. They obtained a much higher activation barrier (24.4 kcal/mol), a very endothermic product (21.7 kcal/mol), and a later TS using QM/MM with a QM region consisting of 77 atoms and optimized using B3LYP/631+G(d). Their results are difficult to compare with ours, because their QM part is considerably smaller (77 atoms) than ours, and the results from the QM-part alone have not be presented. Neither has the procedure for not ending up in different local minima in the MM-part been described. Furthermore, the model of Re et al. has a different protonation state compared to the systems studied by us and by Himo et al.: one of the aspartates of their model is protonated. The protonated residue (PSP-Asp167) corresponds to Asp703 of the Ca2+-ATPase. One of the oxygens of the Asp703 carboxylate group coordinates to Mg2+, the other is exposed to the aqueous environment, so it is safe to assume that this

residue is ionized in the Ca2+-ATPase. How this protonation can affect the energetics has not been studied here. Finally, the TS of Re et al. has not been optimized, leaving us with TSapproximations in the two-dimensional (2D)-energy profile. These many methodological differences make it impossible to explain the different results obtained by Re et al. and by us.

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CONCLUSIONS In the present work we used the quantum cluster approach to investigate the hydrolysis of E2P, the second Ca2+-ATPase phosphoenzyme. The model was based on the X-ray structure 1WPG, which models the product state of E2P hydrolysis. The hydrolysis reaction is found to occur via a nucleophilic attack by a water molecule, with Glu183 acting as a base in the product state. Important interactions in the reaction mechanism are those between the attacking water molecule and Glu183, Thr625, and Thr181 and between Lys684 and the bridging oxygen of the phosphorylated Asp351. The latter strengthens as the reaction proceeds and stabilizes the negative charge on the leaving group. The reaction is catalyzed by a preorganized active site, where the main movement of the nonreacting groups is a rotation of Thr625 toward the phosphate group. The energy barrier of E2P dephosphorylation was calculated to be 14.3 kcal/mol, which is consistent with experimental results considering that the E2P ground state has a lower energy than our model of the reactant. The reaction is calculated close to thermoneutral in accordance with its experimentally observed reversibility. The calculations also show that a more exergonic product, which would make the reaction irreversible (activation energy ∼24 kcal/mol), is not reachable in the main model, which includes Gly626. However when Gly626 was substituted for water the reaction was found to proceed to the low lying product with a fairly low barrier. This indicates that an important purpose of the catalytic site structure is to keep the phosphate group shielded from more water molecules. Regarding methodology, solvation, dispersion, and zeropoint energy corrections have been added to all energies. Our calculations show that the correction is 6−7 kcal/mol smaller for the reactant than for the transition and product states with the dispersion corrections being more than twice as large as the sum of the other two contributions (Table 1). In other words, the dispersion correction is important for the energy profile, since it lowers the activation barrier and decreases the reaction endergonicity.

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ASSOCIATED CONTENT

S Supporting Information *

Xyz files of the structures of reactant, TS, and product1a are provided for model1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.E.R.); andreas.barth@ dbb.su.se (A.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Vetenskapsrådet. 9230

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