A Computational Study - American Chemical Society

Article pubs.acs.org/JPCB

Can Arsenates Replace Phosphates in Natural Biochemical Processes? A Computational Study A. K. Jissy† and Ayan Datta*,†,‡ †

School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram, CET Campus, Thiruvananthapuram-695016, Kerala, India ‡ Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur-700032, West Bengal, India S Supporting Information *

ABSTRACT: A bacterial strain, GFAJ-1 was recently proposed to be substituting arsenic for phosphorus to sustain its growth. We have performed theoretical calculations for analyzing this controversial hypothesis by examining the addition of phosphate to ribose and glucose. Dispersion corrected Density Functional Theory (DFT) calculations in small molecules and QM/MM calculations on clusters derived from crystal structure are performed on structures involved in phosphorylation, considering both phosphates and arsenates. The exothermicity as well as the activation barriers for phosphate and arsenate transfer were examined. Quantum mechanical studies reveal that the relative stability of the products decrease marginally with successive substitution of P with As. However, simultaneously, the transition state barriers decrease with P replacement. This indicates that, kinetically, addition of As is more facile. Pseudorotation barriers for the pentavalent intermediates formed during the nucleophilic attack are also analyzed. A monotonic increase in barriers is observed for pseudorotation with the successive replacement of phosphorus with arsenic in methyl-DHP. A glucokinase crystal structure was chosen to construct a model system for QM/MM calculations. Free energy of the reaction (ΔG) reduces by less than 2.0 kcal/ mol and the activation barrier (ΔG‡) decreases by ∼1 kcal/mol on arsenic incorporation. Thus, both DFT and QM/MM calculations show that arsenic can readily substitute phosphorus in key biomolecules. Secondary kinetic isotope effects for phosphorylation mechanism obtained by QM/MM calculations are also reported. The solvent kinetic isotopic effects (SKIE) for ATP and ATP (As) are calculated to be 5.81 and 4.73, respectively. A difference of ∼1.0 in SKIE suggests that it should be possible to experimentally determine the As−phosphorylation process.

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INTRODUCTION In 2011, Simon et al. reported the discovery of an unusual microbe strain GFAJ-1, which could substitute arsenic for phosphorus and vary the elemental composition of its basic biomolecules.1 The possibility of an alternative, arsenic-based life form, an element which is known to be toxic, created much interest, but also raised concerns over the validity of this hypothesis.2−10 Arsenic, situated directly below phosphorus, shares many key chemical properties with this element.11 Arsenates (HAsO4−2) and phosphates (HPO4−2) are abundant on earth, have nearly identical pKa values,12 share similar thermochemical radii that differ by only 4%,13 and form analogous esters. Thermochemical radii are the ionic radii of polyatomic ions, such as carbonate, which is estimated from the lattice energies of their compounds by using the Kapustinskii equation.14 Irrespective of the similarities, phosphate is essential for life and arsenate is toxic. However, there are several reasons for nature selecting phosphorus and not arsenic for synthesizing essential biomolecules. Compared to phosphate esters, arsenate esters are highly unstable in water, and the hydrolysis rates of arsenate esters are orders of magnitude greater than those of the corresponding phosphates.15−19 Higher reactivity with water would also lead to problems for the stability of the DNA/ RNA containing arsenate-linked nucleic acids. In addition, a © 2013 American Chemical Society

multitude of metabolic reactions would have to substitute arsenate for phosphate. Thus the corresponding enzymes acting on phosphate substrates would need to be mutated as the natural enzymes selected by nature are generally inhibited by the arsenate substrates. Mládek et al. performed quantum chemical calculations to investigate the geometry and electronic properties of the arsenate analogue of the DNA backbone.20 They concluded that arsenate anions may behave as a perfect substitute for phosphate in DNA backbone. However, the incorporation of phosphates in biosystems begins from the phosphate transfer from ATP to different molecules. Thus, by applying high-level quantum chemical calculations, we have analyzed the incorporation of phosphates into the sugar moiety, which finally becomes a part of biomolecules. In biosystems, D-ribose is one sugar which must be phosphorylated by the cell before it can be used. Ribokinase is the enzyme which catalyzes this reaction by converting D-ribose to D-ribose-5-phosphate. Once converted, D-ribose-5-phosphate is available for the synthesis of Received: March 25, 2013 Revised: June 17, 2013 Published: June 21, 2013 8340

dx.doi.org/10.1021/jp402917q | J. Phys. Chem. B 2013, 117, 8340−8346

The Journal of Physical Chemistry B

Article

Scheme 1. Addition of Phosphoric Acid, Pyrophosphoric Acid, and DHP to β-D-deoxyribose (X = P, As)

than the bond-breaking site results in a change in the rate of the reaction, it is known as secondary isotopic effect and can provide information about the role of motion of functional groups/weakly bound molecules that are placed physically away from the bond making/bond breaking site.24 We have looked into the kinetic solvent isotopic effect on arseny-/phosphorylation. Solvent isotopic effect is the effect of isotopic exchange on the reaction rate even when the atom involved is of solvent molecules, rather than of a reactant that has been exchanged for one of the isotopes. The deuteration of the three water molecules which are existing in the glucokinase crystal structure and studied at the QM level in the QM/MM calculations are considered. These three H2O/D2O molecules are in a combined motion in the transition state and are analyzed to investigate the secondary kinetic isotopic effect of water on phosphorylation.

the nucleotides, tryptophan and histidine, or can be used in the pentose phosphate pathway.21 ATP + D‐ribose → ADP + D‐ribose‐5‐phosphate

Thus, ATP and D-ribose are the two substrates leading to the formation of ADP and D-ribose-5-phosphate. As D-ribose-5phosphate is involved in the production of many important biomolecules, it is instructive to study the phosphorylation of sugar with subsequent replacement of phosphorus by its analogue, arsenic. We have thus, modeled our system based on this reaction (Scheme 1). We have considered phosphoric acid, pyrophosphoric acid, diphosphono hydrogen phosphate (DHP), and their arsenic analogues as substrates for addition of phosphate to ribose. The interaction energies and activation barriers for phosphate and arsenate transfer are compared to determine the feasibility of arsenic incorporation. Combined quantum mechanical/molecular mechanical (QM/MM) methods have become an important tool of choice for modeling enzyme catalyzed reactions and predicting the free energies and activation barriers with high chemical accuracy.22 The substrate(s) and only the relevant (conserved) residues in the active site are treated quantum mechanically. The rest of the protein is described at the empirical molecular mechanics level. This makes it possible to treat large enzyme systems as the computational cost is considerably lowered. We have constructed a model system from a reported glucokinase crystal structure, for understanding phosphorylation of glucose and ribose. QM/MM calculations are performed on the model system considering ATP and arsenylated ATP as phosphate/ arsenate donor. One powerful kinetic tool for understanding enzyme reactions is the isotope effect.23 Isotope effects provide information about the nature of the transition state in the rate determining step. If the isotopic substitution at a site other

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COMPUTATIONAL METHODS All the quantum mechanical calculations were performed using the Truhlar’s M06−2X version of DFT which has been remarkably successful in describing dispersion in a variety of molecular systems.25−28 M06−2X functional is a highly parametrized metahybrid method which has shown to be adequate for the study of an intermediate range of electron− electron interactions involved in enzyme catalysis.29 The tripleζ 6-311+G(d,p) basis set was employed.30 Additional frequency calculations were performed to verify all positive vibrational normal modes for the ground state structures and a single negative frequency for transition state (TS) structures. Solvent calculations for all structures were also performed in water using the Polarizable Continuum model (PCM).31 In the PCM model, the solute is embedded in a cavity free of any solvent molecule, and the solvent (water) is considered to be a continuum outside the cavity. The cavity is determined from 8341



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substrate. The TS structures for phosphory-(arseny-)lation of ribose were also determined. Figure 1 shows the TS structure

the molecular surface, as constructed from the scaled van der Waals radii of the constituent atoms. The exothermicity of phosphory-/arseny-lation is evaluated as ΔG298K,1atm = G P 298K,1atm − G R 298K,1atm

where ΔG is the free energy change, GP is the sum of the free energies of the optimized products, and GR is the sum of the free energies of the optimized reactants. The crystal structure (PDB id: 3ID8) of human pancreatic glucokinase crystallized with glucose and AMP-PNP was used to construct the model system for QM/MM computations.32 The model reaction system was simplified to include sugar, ATP, Mg2+, aspartic acid, and lysine. The two most important amino acid residues were included. All other residues were removed. The unsatisfied valencies are terminated by H-atoms. QM/MM calculations were performed using the two-layer ONIOM method33 incorporated in Gaussian 09.34 QM/MM calculations on reactions in enzymes with density functional theory have been successful in reproducing experimental results.35 The QM and the MM regions are calculated at B3LYP/6-31G(d) and AMBER, respectively.36

Figure 1. Transition state structure for transfer of phosphate from methyl-DHP to β-D-deoxyribose at M06−2X/6-311+G(d,p).

for phosphate transfer from methyl-DHP to ribose. The TS corresponds to the transfer of proton from sugar to phosphate. Subsequently the system undergoes an internal proton transfer which leads to the rupture of the phosphoester bond and attack of phosphate on the sugar hydroxyl group. Except for DHP, arsenic substitution results in a steep decrease in the activation barriers. Phosphorylation of methyl-DHP involves crossing a barrier of 35.7 kcal/mol, whereas for 3As-methyl-DHP the barrier decreases by ∼15.0 kcal/mol. We closely looked at the TS geometries for the reaction with methyl-DHP and its allarsenic analogue. The H-bond lengths in the TS for methylDHP are 1.87 Å, 1.97 Å, 2.9 Å, and 2.2 Å which are longer then the hydrogen bonds observed in the TS for arsenylated methylDHP (1.82 Å, 2.0 Å, 2.2 Å, and 1.9 Å). Thus, the transition states are more stabilized by H-bonding interactions in 3Asmethyl-DHP as compared to methyl-DHP. Similarly, when pyrophosphoric acid acts as a donor, the energy barriers are 10−15 kcal/mol lower for the reactions involving arsenic substrates as arsenate donors. Lopez at al. have shown that, during nucleophilic attack on phosphate esters, the reaction further proceeds to the formation of stable neutral phosphorane intermediates.37,38 The intermediates involve a pentavalent P/As, simultaneously coordinated to the oxygen atoms of beta phosphate as well as of sugar. We have determined the structures of these intermediates for the reaction of methyl-DHP and its arsenic analogues. As expected from the atomic radii, both the equatorial (eq) and axial (ax) bond lengths increase on moving from phosphorus to arsenic substrate. The equatorial bond length increases from 1.66 (P) to ∼1.78 Å (As). Similarly the axial bond lengths increase from 1.60 to 1.62 Å (P) to 1.74−1.76 Å (As). The corresponding differences between axial and equatorial distances (ax − eq) are observed to decrease from 0.052 Å to 0.03 Å on replacing phosphorus with arsenic. Such a trend is in harmony with the results of Moc and Morokuma.39,40 The lifetime of the phosphorane intermediate depends on the nature of ligands surrounding the phosphorus center, the protonation state, and its interaction with solvent, metal ions, and functional groups that occur in a macromolecular environment. If the phosphorane intermediate is sufficiently long-lived, it can undergo pseudorotation.41,42 Pseudorotation is a conformational rearrangement which involves a square pyramidal transition state finally resulting in a bipyramidal structure with an apparent rotation of the original trigonal

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RESULTS AND DISCUSSION The interaction energy analyses of the reactions depicted in Scheme 1 are tabulated in Table 1. In order to mimic ATP, the Table 1. Energetics (Gas Phase) of β-D-Deoxyribose Phosphorylation by Phosphoric Acid, Pyrophosphoric Acid, DHP, and Their Arsenic Analogues along with the Free Energy Barrier for the Reactions at M06-2X/6-311+G(d,p) Level molecule

ΔE (kcal/ mol)

ΔE‡ (kcal/ mol)

ΔG (kcal/ mol)

ΔG‡ (kcal/ mol)

H3PO4 H3AsO4 H4P2O7 H4PAsO7 H4As2O7 H5P3O10 H5P2AsO10 H5PAs2O10 H5As3O10 CH3H4P3O10 CH3H4P2AsO10 CH3H4PAs2O10 CH3H4As3O10

−13.0 −11.6 −7.3 −5.4 −5.1 −14.3 −10.5 −11.0 −8.8 −16.4 −12.4 −12.4 −10.4

34.0 27.7 18.6 25.5 26.5 27.3 27.0 34.0 20.6 20.9 20.7

−9.8 −7.5 −7.4 −5.1 −6.0 −12.5 −8.8 −9.9 −7.3 −15.1 −11.0 −11.3 −9.7

33.21 25.2 18.8 26.1 25.2 26.1 25.7 35.7 20.4 20.8 20.5

methyl derivative of DHP was also considered, with the adenosine part replaced by methyl group. For all the systems, gas phase calculations show that the addition of an arsenate to ribose is energetically slightly less favorable (∼2.0 kcal/mol −6.0 kcal/mol) as compared to phosphate addition. The Hbond distances in phosphorylated and arsenylated ribose are 1.88 Å, 2.67 Å, 2.59 Å and 1.89 Å, 2.83 Å, 2.79 Å, respectively. Thus, the product formed by phosphate transfer is more stable as compared to the arsenylated sugar. However, if we look at the reactants, that is, DHP and its arsenic analogue with three arsenic atoms, the H-bond distances are 1.98 Å, 2.01 Å, 2.29 Å and 1.93 Å, 1.93 Å, 2.23 Å, respectively. The higher stability of products, with the additional lower stability of reactants, makes the reaction energetically more feasible for an all-phosphorus 8342



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Table 3. Energetics (Gas Phase and Solvent = H2O) of β-DDeoxyribose Phosphorylation by Oxyanions of Phosphoric Acid, Pyrophosphoric Acid, DHP, and Their Arsenic Analogues at M06-2X/6-311+G(d,p) Level

bipyramid. The transition barriers for pseudorotation increase monotonically with the successive replacement of phosphorus with arsenic in methyl-DHP. The transition barriers of pseudorotation for 2As-, As-, and 3P- methyl-DHP are 4.4, 7.9, and 15.5 kcal/mol, respectively. Further, the reaction proceeds to the cleavage of O−P (between alpha and beta phosphate) bond through a transition state with a proton oriented toward the leaving group so as to protonate it. The solvent calculations (Table 2) also show a trend similar to the gas phase calculations. The products become relatively Table 2. Energetics (Solvent = H2O; PCM Model) of β-DDeoxyribose Phosphorylation by Phosphoric Acid, Pyrophosphoric Acid, DHP, and Their Arsenic Analogues along with the Free Energy Barrier for the Reaction at M062X/6-311+G(d,p) Level molecule

ΔE (kcal/ mol)

ΔE‡ (kcal/ mol)

ΔG (kcal/ mol)

ΔG‡ (kcal/ mol)

H3PO4 H3AsO4 H4P2O7 H4PAsO7 H4As2O7 H5P3O10 H5P2AsO10 H5PAs2O10 H5As3O10 CH3H4P3O10 CH3H4P2AsO10 CH3H4PAs2O10 CH3H4As3O10

−6.9 −6.1 −7.7 −6.2 −6.0 −8.7 −6.0 −6.1 −4.2 −10.3 −7.4 −7.3 −5.7

34.6 25.2 20.6 25.5 24.6 25.3 24.7 33.6 18.3 23.1 17.7

−5.1 −3.3 −8.4 −6.2 −6.5 −8.5 −5.7 −6.2 −4.5 −10.0 −7.3 −6.6 −5.9

33.2 23.4 19.5 24.2 22.4 24.2 21.6 35.7 18.7 22.1 18.2

a

molecule

ΔE (kcal/ mol)

ΔG (kcal/ mol)

ΔEa (kcal/ mol)

ΔGa (kcal/ mol)

H2PO4− H2AsO4− H2P2O72− H2PAsO72− H2As2O72− H2P3O103− H2P2AsO103− H2PAs2O103− H2As3O103− CH4P3O103− CH4P2AsO103− CH4PAs2O103− CH4As3O103−

−23.2 −21.2 −70.0 −66.8 −65.6 −138.0 −132.9 −130.8 −129.3 −131.2 −125.9 −124.6 −126.9

−20.1 −18.4 −71.0 −67.7 −66.2 −135.5 −130.2 −128.3 −126.6 −130.4 −124.8 −123.8 −124.8

−9.5 −8.9 +1.1 +2.2 +2.8 −10.3 −9.0 −8.3 −8.5 −7.8 −6.5 −5.5 −7.5

−6.8 −6.4 −0.2 +1.0 +1.7 −8.0 −7.1 −6.9 −6.3 −7.4 −6.5 −4.7 −6.3

PCM calculations.

bond lengths are longer in solvent compared to in gas phase. The hydrogen bond distances in ribose-phosphate and ribosearsenate in gas phase are 2.43 Å and 2.58 Å, respectively, whereas the hydrogen bond distances are 2.58 Å and 2.73 Å in water for phosphorylated and arsenylated ribose, respectively. Hence, the stabilization of the products by hydrogen bonds is greater in gas phase as compared to in water. In order to attain a better picture of the microenvironment where in vivo phosphorylation takes place, we looked at kinases, the group of enzymes involved in transfer of phosphoryl group from a phosphate donor (usually ATP) to a phosphate acceptor. Ribokinase (RK)-like kinases are the enzymes involved in the phosphorylation of ribose and glucose.43 Despite the diversity observed in RK-like kinases, the substrate binding pocket has similar overall geometry.44 The phosphoryl transfer involves the deprotonation of sugarhydroxyl group by a catalytic base, mostly Asp residue. The oxygen atom then attacks the γ phosphate group of ATP. The kinase members also utilize a magnesium ion and an additional electrostatic interaction between a lysine (or equivalent arginine) and β-phosphate of ATP. Thus, considering the conserved residues we have modeled a system including sugar, ATP, Mg2+, aspartic acid, and lysine for QM/MM computations from a glucokinase crystal structure (PDB id: 3ID8). Phosphorylation of both glucose and ribose were studied. The quantum mechanics region includes the β-, γ-phosphate group, hydroxymethyl group of sugar, three water molecules, anionic carboxymethyl group of aspartate residue, and the methyleneammonium group of lysine. We understand the importance of taking into account the conformational variability of enzymes. However, in this work we restrict to optimizing the reactants and products by consecutive replacement of phosphorus with arsenic. Our present aim is not to calculate accurate absolute energies, but to understand the effect of incorporation of arsenic on phosphorylation, relatively. In future, a more in-depth study of structures taken from MM and QM/MM molecular dynamics simulations would be performed to get a better understanding of the kinetics of the reaction. Table 4 shows the relative stability of the sugar phosphorylated by ATP and its arsenic analogues. The ΔE and ΔG values calculated by QM/MM computations also follow the same

less stable with subsequent replacement of phosphorus by arsenic. The reactions with phosphorus substrates are more favorable by ∼1.0−5.0 kcal/mol, whereas the activation barriers are much lower for the arsenic substituted reactions by nearly ∼15.0 kcal/mol. Both gas phase and solvent phase calculations reveal that arsenic can be a potential substitute of phosphorus in biomolecules. In neutral solution, as ATP is ionized and exists mostly as ATP4−, we also studied the reaction with the oxyanions of H3PO4, H4P2O7, H5P3O10, CH3H4P3O10, and their arsenic analogues. The energetics for phosphorylation of ribose by the oxyanions is shown in Table 3. In the presence of charged oxygen, the relative stability of the products increases with increase in the length of the phosphate donor. The H-bond distances in H2PO4−, H2P2O72−, and H2P3O103− are 2.55 Å, 1.82 Å, and 1.79 Å, respectively. Comparing the reaction with H2P2O72− and H2P3O43−, H-bond of the reactant is 0.03 Å shorter for H2P2O72−, whereas the H-bond length of the product is 0.62 Å shorter for the reaction with H2P3O103−. Hence, the products get more and more stabilized with the increasing chain length of the reacting phosphate. As the Hbond distances are similar in H2P3O103− and its methyl derivative, the interaction energies for these reactions are also nearly the same. In the case of solvated systems, for all substrates, PCM calculations predict a much lower exothermicity for phosphorylation compared with the gas-phase reactions. The charged reactants have a strong tendency to get stabilized due to solvation and therefore are less inclined to lead to the phosphorylated product as compared to gas phase. In addition, in phosphorylated and arsenylated ribose, the H8343



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Table 4. Energetics of Phosphorylation of Glucose and β-DDeoxyribose by ATP and Its Arsenic Analogues (QM/MM: B3LYP/6-31G(d)/AMBER)a molecule

ΔEb

ΔGb

ΔEc

ΔGc

ATP ATP I ATPII ATPIII

−16.4 −11.3 −11.1 −15.8

−4.4 +4.0 +1.7 −7.6

−2.1 −0.1 +1.0 −0.8

+2.3 +6.1 +6.8 +6.7

be +27.1 kcal/mol, about 16.0 kcal/mol higher than the barrier for glucose. Measurement of kinetic isotopic effects (KIEs) has found widespread use in mechanistic studies of various reaction types. We specifically examine the theoretical solvent kinetic isotopic effects (SKIEs) for protons of the three water molecules residing in the coordination sphere of magnesium. The Arrhenius equation gives the dependence of the rate constant k on the activation energy and temperature

a ATP − Adenine TriPhosphate; ATPI − ATP with one phosphorus replaced by arsenic; ATPII − ATP with two phosphorus replaced by arsenic; ATPIII − ATP with three phosphorus replaced by arsenic. b Glucose. cRibose.

k = A exp( −Ea /kBT )

where k is the rate constant, A the pre-exponential factor, Ea is the activation energy, kB is the Boltzmann constant, and T = 300 K. Previously it was shown that kinetic isotopic effect can be evaluated by using ground- and transition-state vibrational normal modes.46−48 The infrared spectra of the ground and transition states were obtained, and the fundamental frequencies were assigned. Energy, Ea, is evaluated in terms of the vibrational frequencies of the three water molecules in the transition state and the ground state (total of nine νO−H modes). Thus, the light-to-heavy KIE is written in terms of the ratio

trend as revealed by the QM calculations. The overall reaction of phosphate transfer from ATP to glucose is exothermic by −16.4 kcal/mol. About 2.0 kcal/mol reduction in ΔE is observed on substitution of phosphorus by arsenic suggesting that arsenic can be a perfect substitute for phosphorus. As the catalytic activity of a given molecular environment depends on the activation energy barrier, the TS structures were also analyzed. The TS (Figure 2) indicates a partially associative SN2-like mechanism involving TS with a length of axial phosphorus−oxygen bonds2.43 Å and 3.2 Å, and axial arsenic−oxygen bonds2.33 Å and 2.75 Å.45 The TS for the arsenic analogue is more stabilized due to the interaction between the different residues as compared to its phosphorus counterpart. The Mg−O (phosphoryl/arsenyl group) interaction distances are 2.06 Å and 1.99 Å, in the TS for ATP and As-ATP, respectively. The H-bonding distances are also longer in the ATP involved transition state (1.65 Å, 1.81 Å, 1.74 Å, and 2.44 Å) as compared to its arsenic analogue (1.63 Å, 1.65 Å, 1.66 Å, and 2.01 Å). The comparably larger stabilization of the TS lowers the energy barrier by ∼1 kcal/mol. Our model leads to a +11.4 kcal/mol barrier for ATP reaction and a +10.2 kcal/mol activation barrier for the reaction with As-ATP. The activation barrier for phosphorylation of ribose is calculated to

−(E T − EG

− kH e( kBT )H e( kBT )H = = E −(E T − EG − a kD e( kBT )D e( kBT )D Ea

As 1 kcal/mol = 350 cm−1 ⎛ −(ν‡− ν) ⎞ ⎜

⎟

350k T kH e ⎝ B ⎠H = ⎛ ‡ ⎞ −(ν − ν) kD ⎜ ⎟ 350k T e ⎝ B ⎠D

where the subscript H represents water; D represents deuterated water; ν‡ is the sum of the bending, symmetric stretching, and asymmetric stretching modes of the three water molecules in the transition state, and similarly ν is the sum in

Figure 2. (A) Reaction mechanism of glucose phosphorylation by ATP. (B) QM/MM (B3LYP/6-31G(d)/AMBER) optimized structures of reactant, transition state, and product. 8344



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the ground state. As kBT = 0.6 kcal/mol, the isotopic effect can be determined by the following equation: ⎛

⎛ −(ν ‡− ν) + (ν ‡− ν) ⎞⎞ H D ⎟⎟ ⎟⎟ 350 ⎠⎠

where kH/kD represents the solvent kinetic isotopic effect. The calculated SKIE’s for ATP and ATP (As) are 5.81 and 4.73, respectively. A decrease in SKIE is observed on the substitution of phosphorus with arsenic. The large SKIE values show that phosphate transfer involves a collective motion of the three water molecules in order to phosphorylate the sugar moiety. The isotopic effects therefore suggest that these three H2O molecules are important in the mechanism of phosphory-/ arseny-lation and a difference in SKIE in between P/As processes might be experimentally determined.

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CONCLUSION In summary, we studied the effect of substituting phosphorus with arsenic on the mechanism of sugar phosphorylation. Phosphorylated sugar is involved in the synthesis of vital biomolecules. Although quantum mechanical calculations predict a marginally lower stability for arsenylated ribose, the activation barriers for arsenylation are ∼15.0 kcal/mol lower than the barrier for phosphorylation. QM/MM optimizations also reveal that substitution of phosphorus by arsenic is energetically feasible. Erb et al. followed up the controversial discovery of Wolf Simon and concluded that GFAJ-1 strain is an arsenic-resistant, phosphate-dependent organism. However, they also report abiotic formation of some arsenylated compounds (hexose arsenates) on feeding GFAJ-1 with arsenate.49 The participation of arsenic in in vivo processes would also include other factors, such as reactivity in water and the existence of enzymes which would act on arsenate substrates. However, based on the calculations performed on our model system, we conclude that substitution of phosphorus by arsenic in biomolecules is possible from a structural, electronic, and kinetic point of view. As the water molecules in the coordination sphere of magnesium are involved in phosphate transfer, the secondary kinetic isotopic effects for the water molecules are found to be significant. The SKIE of arsenylation is ∼1.0 lower than that for phosphorylation. We predict that accurate measurement of SKIE in the arsenic based bacterial strain, GFAJ-1, can be an interesting tool to confirm the existence of arsenic incorporation in the cell cycle.

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ASSOCIATED CONTENT

S Supporting Information *

Cartesian coordinates, energies, harmonic frequencies, complete Gaussian 09 references. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Wolfe-Simon, F.; Blum, J. S.; Kulp, T. R.; Gordon, G. W.; Hoeft, S. E.; Pett-Ridge, J.; Stolz, J. F.; Webb, S. M.; Weber, P. K.; Davies, P. C. W.; Anbar, A. D.; Oremland, R. S. A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus. Science 2011, 332, 1163−1166. (2) Benner, S. A. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (3) Borhani, D. W. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (4) Cotner, J. B.; Hall, E. K. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (5) Csabai, I.; Szathmáry, E. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (6) Foster, P. L. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (7) Oehler, S. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (8) Reaves, M. L.; Sinha, S.; Rabinowitz, J. D.; Kruglyak, L.; Redfield, R. J. Absence of Detectable Arsenate in DNA from Arsenate-Grown GFAJ-1 Cells. Science 2012, 337, 470−473. (9) Redfield, R. J. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (10) Schoepp-Cothenet, B.; Nitschke, W.; Barge, L. M.; Ponce, A.; Russell, M. J.; Tsapin, A. I. Comment on “A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus”. Science 2011, 332, 1149. (11) Tawfik, D. S.; Viola, R. E. Arsenate Replacing Phosphate: Alternative Life Chemistries and Ion Promiscuity. Biochemistry 2011, 50, 1128−1134. (12) Larsen, E.; Hansen, S. Separation of arsenic species by ion-pair and ion exchange high performance liquid chromatography. Microchim. Acta 1992, 109, 47−51. (13) Westheimer, F. Why nature chose phosphates. Science 1987, 235, 1173−1178. (14) Burrows, A.; Parsons, A.; Price, G. Chemistry: Introducing Inorganic, Organic and Physical Chemistry; OUP: Oxford, 2009. (15) Baer, C. D.; Edwards, J. O.; Rieger, P. H. Kinetics of the hydrolysis of arsenate(V) triesters. Inorg. Chem. 1981, 20, 905−907. (16) Pratt, A. J. Why Nature Chose Phosphates Over Arsenates. Journal of Cosmology 2010, 13, 3601−3608. (17) Richmond, T.; Johnson, J.; Edwards, J.; Rieger, P. Kinetics of Pyroarsenate Hydrolysis in Aqueous Solution. Aust. J. Chem. 1977, 30, 1187−1194. (18) Dixon, H. B. F. In Adv. Inorg. Chem.; Sykes, A. G., Ed.; Academic Press: London, 1996; Vol. 44, pp 191−227. (19) Fekry, M. I.; Tipton, P. A.; Gates, K. S. Kinetic Consequences of Replacing the Internucleotide Phosphorus Atoms in DNA with Arsenic. ACS Chem. Biol. 2011, 6, 127−130. (20) Mladek, A. t.; Sponer, J. i.; Sumpter, B. G.; Fuentes-Cabrera, M.; Sponer, J. E. On the Geometry and Electronic Structure of the AsDNA Backbone. J. Phys. Chem. Lett. 2011, 2, 389−392. (21) Anderson, A.; Cooper, R. A. The Significance of Ribokinase for Ribose Utilization by Escherichia Coli. Biochim. Biophys. Acta 1969, 177, 163−165. (22) Claeyssens, F.; Harvey, J. N.; Manby, F. R.; Mata, R. A.; Mulholland, A. J.; Ranaghan, K. E.; Schütz, M.; Thiel, S.; Thiel, W.; Werner, H.-J. High-Accuracy Computation of Reaction Barriers in Enzymes. Angew. Chem., Int. Ed. 2006, 45, 6856−6859. (23) Schramm, V. L. Enzymatic Transition States: Thermodynamics, Dynamics and Analogue Design. Arch. Biochem. Biophys. 2005, 433, 13−26. (24) Dahlquist, F. W.; Rand-Meir, T.; Raftery, M. A. Application of Secondary Alpha-Deuterium Kinetic Isotope Effects to Studies of Enzyme Catalysis. Glycoside Hydrolysis by Lysozyme and BetaGlucosidase. Biochemistry 1969, 8, 4214−4221.

⎜⎜ ⎜⎜ kH 0.6 = e⎝ ⎝ kD 1

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.D. and J.A.K. thank UGC and CSIR India for financial assistance. A.D. thanks DST, CSIR and INSA for partial funding. 8345



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(25) Zhao, Y.; Truhlar, D. G. Density Functionals for Noncovalent Interaction Energies of Biological Importance. J. Chem. Theory Comput. 2006, 3, 289−300. (26) Zhao, Y.; Truhlar, D. G. Density Functionals with Broad Applicability in Chemistry. Acc. Chem. Res. 2008, 41, 157−167. (27) Jissy, A. K.; Ashik, U. P. M.; Datta, A. Nucleic Acid G-quartets: Insights into Diverse Patterns and Optical Properties. J. Phys. Chem. C 2011, 115, 12530−12546. (28) Nijamudheen, A.; Jose, D.; Shine, A.; Datta, A. Molecular Balances Based on Aliphatic CH−π and Lone-Pair−π Interactions. J. Phys. Chem. Lett. 2012, 3, 1493−1496. (29) Acosta-Silva, C.; Bertran, J.; Branchadell, V.; Oliva, A. QuantumMechanical Study on the Mechanism of Peptide Bond Formation in the Ribosome. J. Am. Chem. Soc. 2012, 134, 5817−5831. (30) Sherrill, C. D.; Takatani, T.; Hohenstein, E. G. An Assessment of Theoretical Methods for Nonbonded Interactions: Comparison to Complete Basis Set Limit Coupled-Cluster Potential Energy Curves for the Benzene Dimer, the Methane Dimer, Benzene−Methane, and Benzene−H2S. J. Phys. Chem. A 2009, 113, 10146−10159. (31) Miertuš, S.; Scrocco, E.; Tomasi, J. Electrostatic Interaction of a Solute with a Continuum. A Direct Utilizaion of ab initio Molecular Potentials for the Prevision of Solvent Effects. Chem. Phys. 1981, 55, 117−129. (32) Petit, P.; Antoine, M.; Ferry, G.; Boutin, J. A.; Lagarde, A.; Gluais, L.; Vincentelli, R.; Vuillard, L. The Active Conformation of Human Glucokinase is not Altered by Allosteric Activators. Acta Crystallogr., Sect. D 2011, 67, 929−935. (33) Dapprich, S.; Komáromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J. A new ONIOM Implementation in Gaussian98. Part I. The Calculation of Energies, Gradients, Vibrational Frequencies and Electric Field Derivatives. THEOCHEM 1999, 461−462, 1−21. (34) Frisch, M. J. et al. Gaussian 09 (Revision A.1); Gaussian, Inc.; Wallingford, CT, 2009. (35) Friesner, R. A.; Guallar, V. Ab Initio Quantum Chemical and Mixed Quantum Mechanics/Molecular Mechanics (QM/MM) Methods for Studying Enzymatic Catalysis. Annu. Rev. Phys. Chem. 2005, 56, 389−427. (36) Zhang, J.; Li, C.; Shi, T.; Chen, K.; Shen, X.; Jiang, H. Lys169 of Human Glucokinase is a Determinant for Glucose Phosphorylation: Implication for the Atomic Mechanism of Glucokinase Catalysis. PLoS One 2009, 4, e6304. (37) Lopez, X.; Dejaegere, A.; Leclerc, F.; York, D. M.; Karplus, M. Nucleophilic Attack on Phosphate Diesters: A Density Functional Study of In−Line Reactivity in Dianionic, Monoanionic, and Neutral Systems. J. Phys. Chem. B 2006, 110, 11525−11539. (38) Uchimaru, T.; Uebayasi, M.; Hirose, T.; Tsuzuki, S.; Yliniemelä, A.; Tanabe, K.; Taira, K. Electrostatic Interactions That Determine the Rate of Pseudorotation Processes in Oxyphosphorane Intermediates: Implications with Respect to the Roles of Metal Ions in the Enzymatic Cleavage of RNA. J. Org. Chem. 1996, 61, 1599−1608. (39) Moc, J.; Morokuma, K. Ab initio MO Study on the Periodic Trends in Structures and Energies of Hypervalent Compounds: FourCoordinated XH4− And XF4− Anions Containing a Group 15 Central Atom (X = Phosphorus, Arsenic, Antimony, Bismuth). Inorg. Chem. 1994, 33, 551−560. (40) Breidung, J.; Thiel, W. A Systematic ab initio Study of the Group V Trihalides MX3 And Pentahalides MX5 (M = P-Bi, X = F-I). J. Comput. Chem. 1992, 13, 165−176. (41) López, C. S.; Faza, O. N.; de Lera, A. R.; York, D. M. Pseudorotation Barriers of Biological Oxyphosphoranes: A Challenge for Simulations of Ribozyme Catalysis. Chem.Eur. J. 2005, 11, 2081−2093. (42) López, C. S.; Faza, O. N.; Gregersen, B. A.; Lopez, X.; de Lera, A. R.; York, D. M. Pseudorotation of Natural and Chemically Modified Biological Phosphoranes: Implications for RNA Catalysis. ChemPhysChem 2004, 5, 1266−1266. (43) Sigrell, J. A.; Cameron, A. D.; Jones, T. A.; Mowbray, S. L. Structure of Escherichia coli ribokinase in complex with ribose and

dinucleotide determined to 1.8 å resolution: insights into a new family of kinase structures. Structure 1998, 6, 183−193. (44) Dyguda, E.; Szefczyk, B.; Sokalski, W. The Mechanism of Phosphoryl Transfer Reaction and the Role of Active Site Residues on the Basis of Ribokinase-Like Kinases. Int. J. Mol. Sci. 2004, 5, 141−153. (45) Dyguda-Kazimierowicz, E.; Sokalski, W. A.; Leszczyński, J. Nonempirical study of the Phosphorylation Reaction Catalyzed by 4Methyl-5-B-Hydroxyethylthiazole Kinase: Relevance of the Theory of Intermolecular Interactions. J. Mol. Modeling 2007, 13, 839−849. (46) Olson, L. P.; Li, Y.; Houk, K. N.; Kresge, A. J.; Schaad, L. J. Theoretical Analysis of Secondary Kinetic Isotope Effects in C-N Rotation of Amides. J. Am. Chem. Soc. 1995, 117, 2992−2997. (47) Storer, J. W.; Raimondi, L.; Houk, K. N. Theoretical Secondary Kinetic Isotope Effects and the Interpretation of Transition State Geometries. 2. The Diels-Alder Reaction Transition State Geometry. J. Am. Chem. Soc. 1994, 116, 9675−9683. (48) Wiest, O.; Houk, K. N.; Black, K. A.; Thomas, B. Secondary Kinetic Isotope Effects of Diastereotopic Protons in Pericyclic Reactions: A New Mechanistic Probe. J. Am. Chem. Soc. 1995, 117, 8594−8599. (49) Erb, T. J.; Kiefer, P.; Hattendorf, B.; Günther, D.; Vorholt, J. A. GFAJ-1 is an Arsenate-Resistant, Phosphate-Dependent Organism. Science 2012, 337, 467−470.

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