Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors

Accepted Manuscript Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors

Hammad Ali Hassan, Sadaf Rani, Tabeer Fatima, Farooq Ahmad Kiani, Stefan Fischer PII: DOI: Reference:

S0301-4622(17)30227-2 doi: 10.1016/j.bpc.2017.08.003 BIOCHE 6035

To appear in:

Biophysical Chemistry

Received date: Revised date: Accepted date:

2 June 2017 31 July 2017 13 August 2017

Please cite this article as: Hammad Ali Hassan, Sadaf Rani, Tabeer Fatima, Farooq Ahmad Kiani, Stefan Fischer , Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors, Biophysical Chemistry (2017), doi: 10.1016/j.bpc.2017.08.003

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ACCEPTED MANUSCRIPT 1

Effect of Protonation on the Mechanism of Phosphate Monoester Hydrolysis and Comparison with the Hydrolysis of Nucleoside Triphosphate in Biomolecular Motors

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Hammad Ali Hassan,a Sadaf Rani,a Tabeer Fatima,a Farooq Ahmad Kiani,a,b* Stefan Fischer,c a Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), 44000 Islamabad, Pakistan. b Department of Physiology and Biophysics, Boston University School of Medicine, 72 East Concord Street, 02118-2526 Boston, Massachusetts c

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Interdisciplinary Center for Scientific Computing (IWR), University of Heidelberg, Im Neuenheimer Feld 205, D-69120 Heidelberg, Germany.

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KEYWORDS: Phosphate monoester hydrolysis, Reaction mechanism, Phosphoryl transfer reactions, Computational Biochemistry

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ACCEPTED MANUSCRIPT 2 ABSTRACT:

Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the Oa-H bond of the attacking water molecule, and the P-Ol bond of the substrate (Oa and Ol stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of

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Oa-H and P-Ol bonds occurs simultaneously, whereas in the sequential mechanism, the Oa-H and P-Ol

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bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and

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deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and

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deprotonated cases, the breaking of the water Oa-H bond occurs before the breaking of the P-Ol bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as

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semi-concurrent. The overall energy barrier is 40 kcal mol-1 in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water Oa-H bond. This component is lowered from 34 to 25 kcal

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mol-1 by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the P-Ol bond and is not sensitive to protonation. This is consistent with

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previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water Oa-H bond and to

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have little effect on the P-Ol bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the P-Ol bond could not be found here. This is because the leaving group in phosphate

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monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the β- and γphosphates had been shown to promote a mechanism where the P-Ol bond breaks before the Oa-H bond

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does. We also point out that the charge shift due to P-Ol bond breaking during sequential ATP hydrolysis in bio-molecular motors onsets the week unbinding of hydrolysis product that finally leads to the product release during power stroke.

ACCEPTED MANUSCRIPT 3 Introduction The hydrolysis of phosphate containing compounds is a key chemical reaction in biology.[1] Many computational quantum mechanical studies have been carried out to understand the hydrolysis mechanism of compounds with a P-O-R linkage, where R can be for

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example an alkyl group (e.g., phosphate monoester) or a nucleotide diphosphate (e.g., ATP [2,3])

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However, the details of this deceptively simple reaction are to-date not completely understood

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[4]. The hydrolysis of phosphate has been computationally studied with a variety of substrates and quantum methods such as Hartree-Fock, density functional theory and semi-empirical

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quantum methods. These studies have focused on the hydrolysis mechanism in biological

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systems as diverse as the DNA cleaving enzyme EcoRV [5], ATP-consuming biomolecular motors [6-9] and the signaling enzyme RAS-GAP [10-13]. In addition, quantum chemical studies

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on phosphate monoesters [14,15], diesters [15-17], triesters [18], nucleoside diphosphate [19]

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and nucleoside triphosphates [20,21] have been performed. The results are sometimes inconclusive [22] and need to be re-evaluated with a reliable and systematic quantum chemical

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approach[23].

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Phosphate hydrolysis requires the nucleophilic attack of a water (called here Wa) onto the

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phosphorus atom and the breaking of two strong bonds, i.e., the P-Ol bond of the P-Ol-R linkage and the Oa-H bond of the attacking water molecule (Ol and Oa designate the oxygen atoms of the leaving and the attacking groups), see Figure 1. If the P-Ol and Oa-H bonds break simultaneously, the mechanism is said here to be concurrent (Figure 1AB≠C). If the P–Ol and Oa-H bonds break one after the other and these events are separated by a stable intermediate, the mechanism is referred to as sequential [21,24]. In case the Oa-H bond breaks after the P–Ol bond (Figure 1, AG≠HI≠C), with a marked metaphosphate intermediate (Figure 1H),

ACCEPTED MANUSCRIPT 4 this is referred to here as sequential type P-O mechanism. Recent studies suggest that, for example, myosin [8,24] and other bio-molecular motors [25-27] hydrolyze ATP using this mechanism. Alternatively, the case where the P–Ol bond breaks after the Oa-H bond (Figure 1 AD≠EF≠C) is referred to here as sequential type O-H mechanism. Recent quantum

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chemical studies of the hydrolysis of the phosphate monoester dianion [14,28] showed that when

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the attacking water molecule is positioned apically to the phosphate (i.e, in line with the P-Ol

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bond), then the hydrolysis occurs via this sequential type O-H mechanism.

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Phosphate monoester hydrolysis is one of the slowest reactions in the absence of enzymes,[29,30] and the enzymes that catalyze the hydrolysis of phosphate monoester are among

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the catalytically most efficient enzymes.[31,32] Methyl phosphate monoester is the smallest

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model substrate that can undergo hydrolysis of its P-O-C linkage. The CH3 group in methyl

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phosphate is an extremely poor leaving group. It can be deduced from Linear Free Energy Relationships (LFER) plots[29] Kinetic Isotope Effect (KIE),[33,34] and recent computational

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studies[35] that the transition state to the hydrolysis of methyl phosphate dianion is likely to have more concurrent character as compared to that of an efficient leaving group. Computational

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studies[34] showed that during the hydrolysis of phosphate monoester dianion, a post-hydrolysis

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shallow intermediate is formed just after the pentaphosphorane transition state. We recently showed that varying the protonation of a methyl-triphosphate substrate significantly affects the mechanism of hydrolysis [21]. For instance, the energy barrier for the breaking of the P-Ol bond (which is highly resistant to spontaneous hydrolysis, [36]) is significantly lowered by protonating the leaving αβ-diphosphate group ([37], Figure 2A). Depending on how the protons were added to the methyl triphosphate, the energetically most favorable mechanism changed from concurrent to sequential. The purpose of the present study

ACCEPTED MANUSCRIPT 5 is to investigate how protonation might affect the hydrolysis mechanism in the case of phosphate monoester. Methyl phosphate monoester has only one phosphate protonation site (Figure 2B) thus the hydrolysis of deprotonated methyl-phosphate dianion is compared here to hydrolysis of the protonated methyl-phosphate anion.

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We find that in both the deprotonated as well as protonated methyl phosphate, the Oa-H

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bond breaks before the P-Ol bond (Figure 1, AD≠EF≠C). However, the intermediate conformation (Figure 1E) has nearly the same energy as the preceding transition state (Figure

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1D), so that the mechanism can be considered to be ―semi-concurrent‖ of type O-H. Protonation lowers the overall barrier of the reaction. We explain this effect, and discuss how it compares to

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the effects of protonation that were found for methyl-triphosphate.

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Methods

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Methyl phosphate was set-up with the attacking water molecule positioned apically

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relative to the phosphate group, and three additional waters forming a minimal solvation shell and making hydrogen-bonds to the four oxygen atoms of the phosphate group. Attention was

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paid so that this hydrogen-bond network is the same in the deprotonated and protonated reactant

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structures (compare Figure 3A and 4A), to insure that observed differences in the hydrolysis mechanism are due to the protonation and not due to the influence of having different water shells. The geometries of the stationary points involved in the reaction were optimized at M06-2X/6-31+G* level of theory using Gaussian 09 program package [38]. M06-2X is a dispersion-corrected density functional[39] and is a method of choice in recent quantum chemical studies aimed at elucidating the hydrolysis mechanism of phosphate monoesters [23][14] Initial guesses of the transitions state structures were optimized by fixing suitable

ACCEPTED MANUSCRIPT 6 internal coordinates. Resulting geometries were subjected to unconstrained optimization with a tight convergence criterion. Hessian was calculated at each step of transition state optimization. Frequency calculations confirmed that all optimized saddle points are of first order. Single point energies were calculated at M06-2X/6-311+G** level of theory. Energies reported here are M06-

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2X/6-311+G**//M06-2X/6-31+G*+ZPE energies relative to the reactant (where ZPE stands for

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zero-point energy). The geometry and the energy of Post-Transition State 1, the structure

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immediately after the Transition State 1, are energetically close to the Transition State 1 (Table 1). Therefore, instead of only giving relative energies, we also specify error ranges within

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relative energy values (Table 1, and Figures 3 and 4).

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Results

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a) Hydrolysis reaction of the phosphate monoester dianion

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The hydrolysis reaction of the unprotonated phosphate monoester (dianion) is shown in

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Figure 3. The reaction has the sequential type O-H mechanism (AD≠EF≠C, Figure 1). The corresponding energy profile is shown in Figure 5. The attacking water molecule

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(Wa) undergoes the Oa-H bond scission to transfer its proton to the phosphate oxygen, while

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its OH- hydroxyl makes a nucleophilic attack onto the phosphorus atom (Figure 3A3B). The energy cost of this step is 33.6±0.8 kcal mol-1. In this saddle point of the energy surface, the transferred proton is still shared by oxygens Oa and OP (TS1, Figure 3B). Full transfer of this proton to OP results in a trigonal bipyramidal structure (Figure 3C). The energy of that shallow local minimum is 33.8±0.7 kcal mol-1 relative to the reactant. This is so close to the energy of TS1, that this structure cannot be considered to be a stable intermediate of the reaction (see the plateau in Figure 5). Thus, it is called here

ACCEPTED MANUSCRIPT 7 Post-Transition-State 1 (PTS 1). From that stage, the P-Ol bond breaks as Transition State 2 is crossed (E = 40.8±1.9 kcal mol-1, Figure 3D). The final hydrolysis product energy is 0.9 ±0.7 kcal mol-1 above the reactant state (Figure 3E). The noticeable energy plateau in Figure 5 allows decomposing the overall energy barrier

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of 40.8 kcal mol-1 into two contributions: i) The 33.8 kcal mol-1 cost of proton

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transfer/water-attack equivalent to 83% (or ~5/6th) of the overall barrier, and ii) the 7.0 kcal mol-1 cost of breaking the P-Ol bond in the bipyramidal PTS1, equivalent to 17%

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(~1/6th) of the overall barrier. Due to the absence of a clear energy minimum in the PTS1 intermediate (Figure 1E) along the reaction, this mechanism cannot be called sequential, but

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might be categorized as ―semi-concurrent‖ of type O-H.

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b) Hydrolysis reaction of the phosphate monoester anion

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The hydrolysis of the protonated phosphate-monoester (mono-anion) is displayed in Figure 4.

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The reaction proceeds in the same way as for the unprotonated case described above, namely via a semi-concurrent mechanism of type O-H. The structure of each individual stage are also

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very similar to the unprotonated case (compare each panel in Figure 4 to the corresponding panel in Figure 3). The energy profiles can be compared in Figure 5. As observed above for the unprotonated case, the energy makes a plateau between Transition-State-1 and the Post— Transition—State-1 (PTS1). Again, the energy minimum of PTS1 is too shallow to be considered as a stable intermediate. This allows the same decomposition of the overall energy barrier as described above, i.e., into contributions i) from the proton transfer/water-attack versus ii) from the P-Ol breaking. Interestingly, the energetic cost of

ACCEPTED MANUSCRIPT 8 the P-Ol breaking step (33.7 - 25.0 = 8.7 kcal mol-1) is very similar to the value obtained in the unprotonatd case (7.0 kcal/mol, see above). In contrast the cost of the protontransfer/water-attack step is lower by ∆E = -8.2 kcal mol-1 in the protonated than in the unprotonated case (compare 25.4 and 33.6 kcal mol-1). This accounts for most of the

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lowering of the overall barrier ∆E = -7.1 kcal mol-1 (i.e., from 40.8 to 33.7 kcal mol-1) due to

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protonation. Thus, this shows that the main effect of the protonation is to lower the cost of

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the first step of the reaction, namely of proton-transfer/water-attack.

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Discussion and Conclusions

The present results show that the hydrolysis of hydrated methyl phosphate in the

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protonated as well as the deprotonated case has a mechanism that is semi-concurrent of type

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O-H, i.e., the Oa-H bond breaks before the P-Ol bond, but there is no distinctly stable

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intermediate between the two steps.

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a) The effect of Protonation

Protonating the methyl phosphate does not affect the mechanism, but lowers the overall

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barrier from 40.8 to 33.7 kcal mol-1 by making the water-attack easier during the early phase of the reaction. The initial activation phase OaH- + HPO4-CH3

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H2PO52-CH3≠

(protonated case)

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HPO53-CH3≠

(unprotonated case),

is easier than in OaH- + PO42-CH3

ACCEPTED MANUSCRIPT 9 because adding a positive charge (the proton) to the phosphate group makes the phosphate a better (i.e., less negative) target for the nucleophilic attack of the negatively charged OaH- hydroxyl. However, this has only a moderate effect on lowering the energy barrier. The reason is

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that proton Ha needs to be transferred from water Wa to the phosphate group quasi-

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H2PO4CH3≠

is less favorable than 

HPO4-CH3≠

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Ha+ + PO42-CH3

(protonated case)

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Ha+ + HPO4-CH3

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simultaneously with the hydroxyl attack on the phosphorus. This activated process,

(unprotonated case),

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because the added proton turns the phosphate group into a poorer proton acceptor. In

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other words, the protonation of methyl-phosphate has two competing effects on the initial

only 8.2 kcal mol-1).

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activation energy, which results in a small net lowering of the energy plateau in Figure 5 (by

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For the remainder of the reaction (i.e., the breaking of the P-Ol bond), the protonation has ΔE[PTS1  TS2] is 7.0 and 8.7 kcal mol-1 for the

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little effect on the energy barrier:

unprotonated and the protonated cases, respectively. Note that the slightly smaller ΔE[PTS1  TS2] in the unprotonated case might be explained by the advantage of being able to separate two negative charges on PTS1: H2PO42-OCH3≠

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H2PO4--OCH3≠

In the protonated case, this effect is lost:

ACCEPTED MANUSCRIPT 10 H3PO4-OCH3≠

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H3PO4-OCH3≠,

leading to a higher ΔE[PTS1TS2] = 8.7 kcal mol-1.

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b) Choice of reaction coordinates.

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Traditional classification of hydrolysis reactions as associative, dissociative, or concerted[40,41] is based on the position of the transition state or intermediates in the O’Ferrall-Jencks (MOFJ)

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diagram.[42,43] This diagram is a plot between two distances, i.e., a) the distance of attacking

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oxygen atom to the central phosphorus atom (P-Oa distance), and b) the distance of leaving oxygen atom to the central phosphorus atom (P-Ol distance). Recently Marx et. al. used three

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collective variables[44,45], i.e., a) the coordination number of the bridging and β-oxygen atoms

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to the central phosphorus atom, b) the coordination number of all water oxygen atoms to the central phosphorus atom, and c) the coordination number of three phosphate oxygen atoms and

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the bridging oxygen atoms to hydrogen atoms of all water molecules. The free energy surface

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derived from these collective variables is reduced to O’Ferrall-Jencks (MOFJ) plots in two dimensions.[42,45-47]

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We recently pointed out[24] that the two events that mainly contribute towards the energy barrier during the hydrolysis reaction of the phosphate containing species are a) breaking of the Oa-H bond of the attacking water molecule and b) the breaking of P-Oa bond of phosphate containing species. Based on this consideration, a plot between P-Oa and Oa-H distances can be plotted for the hydrolysis of protonated and unprotonated phosphate monoester (Figure 6). Unlike O’FerrallJencks (MOFJ) diagram in which the two reaction coordinates are the distances of attacking and

ACCEPTED MANUSCRIPT 11 leaving oxygen atoms to the same central phosphorus atom, Figure 6 is a plot between the two bonds, i.e., P-Ol and Oa-H bonds that mainly contribute towards the energy barrier of a hydrolysis reaction. Does the proton transfer occur via a proton-wire through water molecules?

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c)

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One question about phosphate hydrolysis is whether some water molecules are relaying the

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proton transfer from the attacking water Wa to the final proton acceptor (here: the phosphate group). Many computational studies have shown that the mechanism of hydrolysis of the POP

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anhydride linkage (e.g., diphosphate and triphosphate hydrolysis) can occur when the proton of

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the attacking water is transferred via a proton-wire composed of one or more water molecules.[8],[24],[48],[20,25,44,49-55] Such solvent assisted hydrolysis mechanism that relays

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protons through other water molecules is not reported in any computational studies of isolated

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compounds containing the P-O-C ester linkage. The most recent studies by Kamerelin et. al.[23] also only reported reaction pathways with direct transfer from one attacking water, Wa, to the

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phosphate. Present work is focused on the effect of protonation on phosphate monoester

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hydrolysis, therefore a comparison between the one-water and two-water mechanisms is beyond

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the scope of the present study.

c) Comparing to the hydrolysis of triphosphate Our recent investigations of the effects of protonation on the hydrolysis of methyl triphosphate [21] have shown that whenever the terminal γ-phosphate is protonated (Figure 2A), this facilitates water attack. For example single protonation in the γ position of triphosphate yielded a semi-concurrent type O-H mechanism with an overall barrier of 32.4 kcal

ACCEPTED MANUSCRIPT 12 mol-1, down from 44 kcal mol-1 in absence of the γ-proton. This is the same mechanism as obtained here with singly protonated phosphate monoester, and nearly exactly the same barrier (32.3 kcal mol-1, Fig. 5) Of the other concurrent reaction pathways that were obtained with various protonation

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patterns of the triphosphate (for example on positions αγ or βγ or αβγ, Figure 2A), all displayed

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the type O-H semi-concurrent mechanism. (i.e., with proton-transfer/water-attack proceeding the breaking of the Pγ-Oβγ bond, but without a distinct energy intermediate between these two

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steps). The explanation is the same as given above for the protonated phosphate monoester: Adding a proton on the γ-phosphate of triphosphate makes it a better target for nucleophilic

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attack by the OaH- hydroxyl. Thus, the effect of protonation obtained here for phosphate

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monoester is the same as had been observed for the γ-phosphate of the triphosphate.

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Conversely, in the same study, it was found that protonation of the leaving group (on either the α- or β-sites of triphosphate, Fig 2A) facilitates the breaking of the P–Ol bond. This is

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because when protons are added to the α- and β-sites, the transfer of electronic charge from the

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cleavable P-O bond to the leaving αβ-diphosphate is facilitated. Thus, it had been possible to calculate several pathways for triphosphate with a sequential mechanism of type PO, involving

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the formation of a distinct metaphosphate intermediate (Figure 1H). Since phosphate monoester only has one phosphate group (Figure 2B), this effect cannot be observed for the hydrolysis of phosphate monoester. Little attention has been paid to calculate the effect of protonation on the mechanism of triphosphate hydrolysis. Akola and Jones optimized the neutral structures of ATP, GTP, methyl triphosphate (MTP), and Mg(MTP).4H2O.[56] Hydrolysis of β,γ-protonated methyl

ACCEPTED MANUSCRIPT 13 triphosphate was studied by Marx et. al.[43] Since β-protonation promotes sequential type P-O mechanism, whereas γ-protonation favors water attack and a concurrent mechanism,[21] the two effects compete each other, and the β,γ-protonated methyl triphosphate undergoes a semiconcurrent sequential type P-O mechanism.[21][57]

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d) Comparison with the ATP hydrolysis in biomolecular motors

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A recent review [58] of the mechanism of ATP hydrolysis in several bio-molecular motors (myosin, Kinesin, F1 ATPase) indicates that, in these motors, ATP hydrolysis follows a

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sequential type P-O mechanism. For example, in the active site of myosin (Figure 7) the

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backbone NH groups of the P-loop residues and the side chains of Lys185 and Asn233 form several hydrogen bond interactions with the α- and β-phosphate of the leaving group. This has a

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similar effect of pulling negative charge towards the αβ-diphosphate as the protonation of these

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group would have [8]. Thus, although an actual protonation of the adenosine triphosphate is not occurring in the active site, the protein alters the mechanism from concurrent (or semi-

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concurrent) to become sequential type P-O. Thereby, the enzyme greatly reduces the overall

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potential energy barrier (down to ~12 kcal mol-1, see ref [59]and ([60] for details).

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e) Sequential hydrolysis is the key to the proper motor function Nath et. al recently proposed a torsional mechanism of the post-hydrolysis changes in biomolecular motors.[67][57,62] According to this mechanism, the changes in catalytic site of a post-hydrolysis motor must follow a sequential pattern. This is because the hydrolysis products Pi- and Mg2+/ADP3- can’t be ejected from the motor in a single step. When ATP is converted into ADP and Pi, the electrostatic interactions of ADP/Pi with the catalytic site of the biomolecular motors are different from that of the ATP reactant. This initiates loose unbinding

ACCEPTED MANUSCRIPT 14 of hydrolysis products from the catalytic site and results in a series of subsequent conformational

changes

in

power

stroke

during

which

hydrolysis

products

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released.[57,62,64-68] Here, we point out a link between the post-hydrolysis torsional mechanism[67] and the

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sequential catalytic mechanism[69] of nucleoside triphosphate hydrolysis in biomolecular

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motors. We pointed out that the concurrent mechanism of hydrolysis in biomolecular motors is energetically unfavorable as it involves simultaneous breaking of O-H bond of water and P-O

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bond of triphosphate. Contrarily, the sequential type P-O mechanism of hydrolysis (Figure 1) that occurs in three steps, i.e., a) the cleavage of P-Ol bond of substrate, b) the breaking of the

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Oa-H bond of water, and c) subsequent rearrangements due to proton transfer is energetically

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favorable.[59] Indeed, during the first step, i.e., the cleavage of P-Ol bond, ATP4- is converted into ADP3-/PO3-. This shifts one negative charge from γ-phosphate to ADP. Subsequently the

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distance between the ADP3- and PO3- moieties increases.[59] These electronic and structural

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changes in going from ATP4- reactant to an ADP3-/PO3- intermediate change the electrostatic interaction of ADP3-/PO3- with the catalytic site of biomolecular motor. Thus charge shift

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changes the electrostatic interactions of ADP3- and PO3- with the binding site and initiates

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small-scaled, early-stage conformational changes in bio-molecular motors, while the Oa-H bond of water is still intact. The next steps are the breaking of the Oa-H bond, and the proton transfer to the γ-phosphate.[59] These small-scaled conformational changes of the weakly bound ADP3/PO3- intermediate are tightly intervened with the large scaled conformational changes in biomolecular motors,[63] so that the products gradually unbind with motor and are finally released during power stroke.

ACCEPTED MANUSCRIPT 15 Conclusion The hydrolysis reaction of the deprotonated and protonated phosphate monoester is computed at the M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory. We find a sequential type OH mechanism in which the Oa-H bond of the attacking water molecule breaks before the breaking of

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P-Ol bond of phosphate monoester. We classify this mechanism as a semi-concurrent of the

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sequential type O-H. This is because the energy and the geometry of the post-hydrolysis intermediate (Post-Transition State 1) are only slightly different from that of the transition state

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(Figures 3 and 4). This mechanism is consistent with the experimental LFER and KIE experiments that show that the hydrolysis mechanism of a phosphate monoester with a poor

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leaving group such as a methyl group has more concurrent characteristics. When the methyl

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phosphate is protonated, the energy barrier to the hydrolysis decreases, but the mechanism of hydrolysis remains unaffected. Our results show that although the hydrolysis of deprotonated

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methyl phosphate is slightly endergonic, the hydrolysis of protonated methyl phosphate is

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exergonic. The rate-limiting step in both protonated and deprotonated methyl phosphate

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hydrolysis is the breaking of the P-Ol bond. The finding that the protonation does not alter the mechanism of hydrolysis in phosphate

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monoester is in contrast to the triphosphate hydrolysis in which protonation at some sites significantly affects the hydrolysis mechanism:[71] Protonation of the leaving phosphate group in triphosphate makes it a good leaving group that favors sequential type P-O mechanism. Nevertheless, the protonation of γ-phosphate facilitates water attack therefore favors concurrent mechanism. This effect is absent in phosphate monoester because it only has one phosphate protonation site.

ACCEPTED MANUSCRIPT 16 For the hydrolysis of triphosphate, biomolecular motors place positively charged residues closer to the leaving diphosphate moiety to transform the hydrolysis reaction from semi-concurrent to sequential. The charge shift due to the P-Ol bond breaking during sequential mechanism changes the electrostatic interaction of ADP3-/PO3- intermediate with the catalytic site of biomolecular

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motors. This change in electrostatic interaction gradually leads to the unbinding and subsequent

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release of ADP/Pi product during power stroke.

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FIGURES

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Figure 1. Some possible mechanisms for the hydrolysis of compounds containing phosphate. In a concurrent mechanism (AB≠C), the nucleophilic attack and the breaking of the P-Ol

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and Oa-H bonds occur simultaneously (≠ denotes a transition state). The sequential mechanism can be of two types: a sequential type O-H mechanism AD≠EF≠C (in which the Oa-H bond breaks before the breaking of the P-Ol bond), or a sequential type P-O mechanism AG≠ HI≠C (in which the P-Ol bond breaks before the Oa-H bond). For a mechanism to be considered as sequential from a kinetic point of view, the intermediate E must be distinctly lower in energy than the surrounding transition states D≠ and F≠ (or H lower than G≠ and I≠ ).

ACCEPTED MANUSCRIPT 18 Otherwise, the mechanism is categorized here as semi-concurrent (of type O-H or P-O, respectively). Interactions shown as  denote a close approach of two atoms. The Oa-H and P-Ol

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bonds that undergo scission during hydrolysis are shown in red.

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Figure 2: A comparison of the methyl triphosphate and methyl phosphate substrates. A) Methyl triphosphate allows several protonation patterns on its three phosphates. B) Methyl monophosphate

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has only two possible phosphate protonation sites.

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Figure 3: Hydrolysis of the phosphate monoester dianion.

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A) The reactant structure consists of CH3-O-PO32- (the P atom is in gold color) and four water

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molecules (the attacking water molecule is labelled as Wa). Hydrogen bonds are shown as broken lines. B) Transition-State 1. Dotted lines indicate the breaking of the Oa-H bond (1.80 A°) and the nucleophilic attack of the Oa-H- onto the P. The water proton Ha is transferred to the phosphate oxygen (HaOp is 0.98 A°). C) Post Transition State 1. Bond Oa-Ha is fully broken (1.85 A°) and bond Ha-OP is fully formed (0.97 A°). D) Transition state 2. The dotted line indicates the breaking of the P-Ol bond E) Product structure: CH3-O- + H2PO4- and three waters. The shown structures are energy optimized stationary points. Energy values are in kcal/mol

ACCEPTED MANUSCRIPT 21 relative to the reactant. Energy values in parenthesis include zero-point energies. Distances are

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given in Ångström.

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Figure 4: Hydroylsis of the phosphate monoester anion. A) The reactant structure consists of CH3-O-PO3H- and four waters (Wa is the attacking water).

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B) Transition state 1. Breaking of the Oa-Ha bond (1.74 Å) and transfer of proton Ha to the

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phosphate oxygen (Ha-OP is 0.99 Å). C) Post-Transition-State 1. The distances Oa-Ha and Ha-OP are 2.00 Å and 0.97 Å, respectively. D) Transition State 2. Breaking of the P-Ol bond. E) Product structure: CH3OH + H2PO4- and three waters. See also caption of Figure 3.

ACCEPTED MANUSCRIPT 23

50

30

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20 10

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Energy (kcal/mol)

40

0 -10

Transition State 1 Post Transition Transition-State 2 State 1

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Reactant

Protonated

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Unprotonated

Product

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Figure 5: Energy levels of phosphate hydrolysis in phosphate monoester

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Energy values relative to the corresponding reactant structure (same as shown in Figures 3 and

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4), error bars are shown for each stationary point.  =unprotonated,  = protonated.

ACCEPTED MANUSCRIPT 24 4 3.5

2.5 2

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1.5 1 0.5

1

1.5

2

2.5

Oa-H Distance (A)

3.5

4

Protonated

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Unprotonated

3

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0

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P-Ol distance (A)

3

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Figure 6: A plot of Oa-H and P-Ol distances.

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Both protonated and unprotonated cases show a sequential type O-H mechanism in which the

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Oa-H bond breaks before the breaking of the P-Oa bond.

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25

Figure 7: The ATP catalytic site of the myosin motor.

The Figure is reproduced with permission from reference [58]. Several hydrogen bond interactions are present between the backbone NH groups of the P-loop (in orange) and the αβ-diphosphate group (which is the leaving group after ATP hydrolysis). Lysine 181 and Asn233 also form hydrogen bonds with the triphosphate moiety. The adenosine attached to the α-phosphate is not shown.

ACCEPTED MANUSCRIPT 26 Table 1. Energy values of structures in the hydrolysis of deprotonated phosphate monoester. M06-2X/6-

M06-2X/6-

M06-2X/6-

M06-2X/6-

Energy

31+G*

31+G*+ZPE

311+G**/M06-

311+G**/M06-

values

2X/6-31+G*

2X/6-

error ranges

0.0

0,0

0,0

0,0

Transition State 1

34.4

33.6

33.5

33.6

Post Transition State 1

34.3

33.8

33.7

Transition State 2

42.7

40.1

Product

1.6

0.6

CR

0.0 33.6±0.8 33.8±0.7

41.5

40.1

40.8±1.9

1.1

0.6

0.9±0.7

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33.8

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Reactant

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31+G*+ZPE

with

ACCEPTED MANUSCRIPT 27 Table 2. Energy values of structures in the hydrolysis of protonated phosphate monoester. M06-2X/6-

M06-2X/6-

M06-2X/6-

M06-2X/6-

Energy

31+G*

31+G*+ZPE

311+G**/M06-

311+G**/M06-

values

2X/6-31+G*

2X/6-

error ranges

0.0

0.0

0.0

0.0

Transition State 1

25.7

24.9

25.9

25.1

Post Transition State 1

24.2

24.7

25.3

Transition State 2

35.3

32.3

Product

-3.5

-3.1

CR

0.0 25.4±0.5 25.0±0.8

35.0

32.0

33.7±1.7

-3.1

-2.7

-3.1±0.4

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Reactant

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31+G*+ZPE

with

ACCEPTED MANUSCRIPT 28 Table 2. Oa-H and P-Ol distances of the protonated and unprotonated methyl phosphate monoesters. Oa-H

P-Ol

Oa-H distance

P-Ol

0.98

1.71

0.98

1.64

1.8

1.78

1.74

1.66

1.85

1.8

2.0

1.77

2.52

2.1

2.2

3.6

3.44

3.23

Reactant

IP

T

Transition State 1

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Post Transition State 1

Intermediate

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2.3

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Product

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ACCEPTED MANUSCRIPT 29 AUTHOR INFORMATION Corresponding Author Farooq Ahmad Kiani Department of Physiology and Biophysics, Boston University School of Medicine, 72 East

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Concord Street, 02118-2526 Boston, Massachusetts. E-mail: [email protected]

Research Center for Modeling and Simulation (RCMS), National University of Sciences and

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Technology, 44000 Islamabad, Pakistan. E-mail: [email protected] Tel: +92-51-

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9085-5733

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Author Contributions

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The manuscript was written through contributions of all authors. / All authors have given

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approval to the final version of the manuscript.

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ACKNOWLEDGMENT ABBREVIATIONS

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ATP, Adenosine triphosphate; ADP, Adenosine diphosphate;

ACCEPTED MANUSCRIPT 30 References

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Graphical abstract

ACCEPTED MANUSCRIPT 36 Highlights:

1. Hydrolysis of Phosphate monoester is a crucial reaction in biology.

2. We report the mechanism of hydrolysis of protonated and dperotnated phosphate

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monoester using density functional computtaions.

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3. The overall energy barrier of phosphate monoester hydrolysis is lowered for the protonated phosphate monoester, however, the mechanism of hydrolysis remains the

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occurs before the breaking of the P-Ol bond.

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same in the protonated and deprotonated cases: the breaking of the water Oa-H bond

4. This is contrary to the triphosphate hydrolysis in which protonation at some sites

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significantly alter the mechanism of hydrolysis.

5. The reported semi-concurrent mechanism of phosphate monoester hydrolysis is

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consistent with experimental findings.