Hydrolysis of aspartic acid phosphoramidate nucleotides: a comparative

quantum chemical study

Servaas Michielssens,*ab Nguyen Tien Trung,ab Matheus Froeyen,c Piet Herdewijn,c

Minh Tho Nguyenab

and Arnout Ceulemansab

Received 25th March 2009, Accepted 18th May 2009

First published as an Advance Article on the web 11th June 2009

DOI: 10.1039/b906020k

L-Aspartic acid has recently been found to be a good leaving group during HIV reverse

transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA.

This showed that L-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine

triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis

of several models for L-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and

G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a

simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic

environment was mimicked by involving two Mg2+ ions and some important active site residues

in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which

is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving

group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier

is lower than the comparable mechanism with methanol as a leaving group. The calculations on

the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the

same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for

L-aspartic-dAMP than for L-malic-dAMP and pyrophosphate.

1. Introduction

Triphosphate has a central role in the metabolism of living

organisms. It is known to be the fuel for numerous biochemical

processes.1,2 Hydrolysis of the high energy P–O bond in tri-

phosphate is coupled to many endothermic reactions in nature.

One of the processes in which triphospate plays a crucial role is

DNA and RNA polymerization. Here the energy stored in

triphosphate is coupled to nucleic acid polymerization. Recently

Adelfinskaya et al.3,4 succeeded in finding new leaving groups

replacing the pyrophosphate leaving group in triphosphate.

Amino acids were coupled with a P–N bond to deoxyadenosine

monophosphate (dAMP) forming phosphoramidates. Especially

the results with L-histidine (L-His) and L-aspartic acid (L-Asp)

were remarkable. Polymerization reactions using L-Asp-dAMP

and L-His-dAMP using HIV reverse transcriptase (HIV RT) were

successful in the synthesis of small strands of DNA. Best results

were obtained with L-Asp-dAMP. This can be rationalized by

looking at the high charge of the latter compound (�3) whichresembles the charge of pyrophosphate (�4) better than L-His.

The search for pyrophosphate alternatives in DNA-

polymerization reactions is important from three perspectives.

The first is in medicinal chemistry. Using nucleotides with a

modified sugar chain termination in DNA polymerization can

be accomplished.5 A major problem with a lot of known

antiviral drugs using this principle, e.g. acyclovir6,7 or azido-

thymidine7 is that they need to be phosphorylated in the cell.

For this they need to pass a kinase pathway which is often

more selective than the viral polymerase. The phosphor-

amidates presented by Adelfinskaya et al.3,4 can be used to

bypass this pathway. This strategy was successfully applied3

for L-Asp-PMEA (phosphomethoxyethyladenine) and sustains

the high expectations of these molecules as antiviral drugs,

provided a better mechanistic-based leaving group can be

found. It is very important that the leaving group is a cellular

metabolite. This means the leaving group is non-toxic and can

be further processed by the cellular metabolism. A second

application is in synthetic biology. These new building blocks

for life can be further developed to take part in an orthogonal

metabolism. Such orthogonal metabolism is necessary to

avoid contamination of artificial life forms in the environment.

Third is a fundamental understanding of polymerase reaction

in nature by use of model components and more specifically to

see if pyrophosphate can be replaced in this reaction.

The high energy of the P–O bond in pyrophosphate has

been traditionally attributed mainly to three factors.8 Firstly

resonance stabilization is less favored in reactants than in

products. This is claimed to be due to the ‘‘opposing resonance

effect’’ caused by competition between an adjacent phosphoryl

groups for the same lone pair on the bridging oxygen atom.

Secondly, electrostatic repulsion is thought to be an important

factor. And finally, solvation effects are shown to be less

a Katholieke Universiteit Leuven, Department of Chemistry andLMCC-Mathematical Modeling and Computational Science Center,B-3001, Leuven, Belgium.E-mail: servaas.michielssens@chem.kuleuven.be

bKatholieke Universiteit Leuven, INPAC Institute of NanoscalePhysics & Chemistry, B-3001, Leuven, Belgium

cKatholieke Universiteit Leuven, Rega Institute for MedicinalResearch, Medicinal Chemistry Laboratory, B-3000, Leuven,Belgium

7274 | Phys. Chem. Chem. Phys., 2009, 11, 7274–7285 This journal is �c the Owner Societies 2009

PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

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favorable for the reactants than for the hydrolysis products.

Recently it has been suggested that the anomeric effect should

replace the resonance effect in this list,8,9 this effect is shown to

contribute for both P–N8 and P–O9 bonds. Mimetic

compounds serving as a replacement for pyrophosphate must

have similar thermodynamic properties. Here we use quantum

chemical methods to calculate the enthalpy difference upon

changing the P–O–CH3 bond in [MeOPO3H]� to P–S or P–N

and changing pyrophosphate to L-asp. We also calculate the

effect of the increasing charge on hydrolysis enthalpy.

Also we investigate the detailed mechanism of the dissociation

path of aspartic acid in hydrolysis reactions. We mimic this

reaction using 4 different models. The first is a simple model

with methyl amine as leaving group. For this simple molecule

we investigate different possible mechanisms. As a second

model we use aspartic acid as a leaving group. Next, we

examine the effect of Mg2+, since it is present in many

enzymes as a catalytic metal. So in the third model we add a

single Mg2+ and in the final model two Mg2+ ions. In all

different models we compare what happens if the dissociating

P–N bond is changed to a P–O bond. In the first model this

corresponds to changing the methyl amine leaving group by

methanol, this gives us an interesting opportunity to compare

with recent theoretical calculations.10,11 In all other models

this corresponds to changing aspartic acid to malic acid, which

gives us an indication of the use of malic acid versus aspartic

acid as a pyrophosphate alternative.

II. Methods

All calculations were performed using the Gaussian 03

package.12 Visualization was done with VMD.13 The structures

were optimized with the B3LYP method14 with DGDZVP15,16

basis set. Surface scans of the different molecules used were

done by manually changing the dihedral angles and comparing

the energies. This was done for L-Asp in different protonation

states. After this mono-methyl-phosphate was added to the 8

lowest conformations and for all of them different conforma-

tions of the monophosphate were scanned. For the thermo-

chemistry calculations G3B3 and G3MP2 methods17,18 were

used. These methods have on average a small absolute

deviation from experiment (0.99 kcal mol�1 for G3B3 and

1.30 kcal mol�1 for G3MP217,18). G3B3 theory uses

geometries and zero-point energy corrections (ZPE) from

B3LYP/6-31G(d) calculations after this higher level single

point calculations are done. Using an interpolation procedure,

energies are obtained comparable to a QCISD(T)/G3 large

computation with much less computing power.19

For the reaction mechanisms the geometry optimization

procedure was the same. Every stationary point was checked

by frequency analysis. ZPE correction using B3LYP were

scaled20 with a factor of 0.9877. The energy of the B3LYP/

DGDZVP optimized structures was corrected doing single

point MP2/6-311++G** calculations on them (indicated as

MP2/6-311++G**//B3LYP/DGDZVP). Single point solvation

corrections using continuum solvent were performed. We used

the PCM21,22 model with Pauling radii; van der Waals radii

were scaled11,23–25 by 1.2. If not explicitly mentionted energies

reported in the text are MP2 energies with solvation

correction.

Molar energies in tables are reported as follows: DEg is the

energy in the gas phase, DGg is the free energy in the gas phase,

DGsol is to solvation energy and DGaq is the free energy in

aqueous solution.

III. Results and discussion

A Influence of bond type and charge on phosphate hydrolysis

The enthalpy for different hydrolysis reactions (see Fig. 1) was

calculated using a high level of theory. The first three reactions

in Table 1 were calculated with both G3B3 and G3MP2,17,18

the difference between the two methods is never larger than

0.5 kcal mol�1. Due to the computational complexity the last

three reactions were only computed at G3MP2 level of theory.

However, as shown in literature18 and confirmed for the first

three reactions, the difference between the two methods

is small.

When analyzing nature’s energy carriers we expect P–N to

be less stable than P–O comparing e.g. free energy for

N0-phosphorylcreatine hydrolysis (�10.3 kcal mol�1) with ATP

hydrolysis (�7.3 kcal mol�1)8 or phosphoester bond in

phophohydroxyamino acids (�6.5 to �9.5 kcal mol) with

phosphoramidate in phosphohistidine(�12 to �14 kcal mol�1).26

This is also confirmed by the calculations.

The most stable conformation used for the reactants is a

conformation with the OH and CH3 group eclipsed. The

hydrolysis reaction with the CH3OH leaving group is

significantly less exothermic than the hydrolysis reactions with

CH3NH2 and CH3SH (see Table 1). This makes the latter

interesting substitutes for pyrophosphate, since reduced bonding

energy might compensate for the loss of e.g. ‘‘opposing

resonance stabilization’’ of L-Asp-dAMP the disadvantage is

that they are less stable towards hydrolysis in solution, which

implies that they could be dissociated before serving as a

substrate for an enzyme. P–O bond length is 1.72 A,

P–N 1.76 A and P–S 2.20 A, suggesting that while CH3NH2

and CH3SH have similar hydrolysis energies CH3SH might

cause a distortion in the active site of enzymes due to the high

difference in bond length.

To compare the results to previous results on triphosphate24,27,28

we also studied the effect of the charge on the reaction. It

is well known that charge is an important factor in the

exothermic nature of triphosphate hydrolysis, see e.g. Strajbl

et al.29 As a model for L-Asp-dAMP10,11,30 we used the molecules

Fig. 1 Different models used for the thermochemistry calculations.

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in Fig. 2. Here we replace the ribose sugar and the base by

hydrogen, since we are mainly interested in the pyrophosphate

substitute. A similar model is used for triphosphate.24,27,28 The

molecule with a charge minus two is protonated on the carboxyl

function of the main chain of aspartic acid. The P–N bond length

decreases with increasing charge, from 1.72 A for the monoanion

over 1.70 A for the dianion to 1.68 A for the trianion. We also

applied the G3MP2 method to recently calculated triphosphate

structures found in literature.27 The trend in our results is similar

to previous studies, see Table 2. For the lower charged species

(�1 and �2) the results agree quantitatively. For the highest

(�3) charge there is a considerable deviation. It should also be

noted that this charge effect is much smaller in solvent.28 What

is important for the molecules with charge �2 and �3 is

the comparison with triphosphate not the absolute value. It is

clear that the trend in hydrolysis enthalpy is analogous to

the trend for triphosphate. Consistent with the weaker

P–N bond these values are systematically more exothermic

than for triphosphate with the corresponding charge. For the

�3 charged molecule, which is expected to be the state at

physiological conditions, the enthalpy for phosphoramidate is

�127.9 kcal mol�1 and for triphosphate�96.4 kcal mol�1. Overall

the results suggest as expected that phosphoramidates with equal

charge are more susceptible to hydrolysis than triphosphate, even

in the absence of the ‘‘opposing resonance stabilization’’, which is

impossible here due to the lack of competing groups for P–N

conjugation.

B Reaction of [MeNHPO3H]� and [MeOPO3H]� + H2O

The simplest model for triphosphate studied is mono-methyl-

phosphate ([MeOPO3H]�).10,11,30 In analogy we use

N-methyl-phosphoramidate ([MeNHPO3H]�) as a simple

model to study the effect of changing the P–O bond in

[MeOPO3H]� to P–N in [MeNHPO3H]�. We calculated four

different hydrolysis reactions and compared those to previous

work10,11,30 and own calculations on the well known hydro-

lysis reaction of [MeOPO3H]�.

Before we start the discussion on the different mechanism

we explain the labels used. All mechanisms are labeled as

follows: X_(ts/int)m_n, the X points to the nature of the

mechanism, then is indicated whether it is a transition state

(ts) or an intermediate (int) and finally m labels the position on

the reaction pathway, and n gives the number of water

molecules involved. There are four different mechanisms

discussed: an associative mechanism (A), a tautomeric

associative mechanism (TA), a tautomeric dissociative

mechanism (TD) and interchange mechanism (Ia).

The first pathway is an associative mechanism (A). In this

well known mechanism water attacks the face of the tetra-

hedron opposite to the leaving group. As a result, a trigonal

bipyramidal intermediate is formed and the configuration on

the phosphorus is inverted. The P–O bond is mainly formed

before the P–N bond is broken. The stationary points for this

reaction are shown in Fig. 3 and energies are reported in

Table 3. In the reactant complex of [MeNHPO3H]� the

attacking water molecule forms a double hydrogen bond with

the N-methyl-phosphoramidate. The P–N bond here is 1.73 A.

In A_ts1_1 a water molecule attacks the phosphoramidate. H1

is transferred to O3 and at the same time the O1–P1 bond

(2.23 A) is partially formed. This TS is a strained four-

membered ring system. It is noted that an attack of a water

dimer could release this ring strain and lower the barrier.31,32

We have explored this in A_ts1_2, the geometry is still

strained, because of the P–O1 distance of 2.19 A and the

O1–P–O3 angle of 93, which are difficult to fit in a six-

membered ring. Instead of lowering, we have a slight increase

of the barrier of 2.2 kcal mol�1 (Table 3). As OH� attacks

the phosphorus a pentacoordinated trigonal bipyramidal

phosphorus with the incoming and leaving group apical is

formed (A_int_1). The next step is the transfer of H2 to N and

the breaking of the P–N bond, this happens in A_ts2_1. The

P–N bond distance is enlarged to 2.18 A and the P–O1

distance reduced to 1.74 A. Using two water molecules in this

transition state gives a remarkable result, A_ts2_1 is lowered

by 7.4 kcal mol�1 with the help of an extra water molecule in

A_ts2_2. This was noticed for the tautomeric dissociative

mechanism of [MeNHPO3H]� by several authors,10,30 but

not for the associative mechanism. As shown in Table 4

for the associative mechanism of [MeOPO3H]� there is no

reduction in the barrier if an extra water molecule is involved.

The crucial parameters seem to be the length of the P–N or

P–O bond. For A_ts2_2 of [MeOPO3H]� the transition state is

very similar (figure not shown), but P–O of the leaving group

is 2.25 A while for A_ts2_1 of P–N the bond distance is 2.0 A.

This is also consistent with the fact that A_ts1_2 is not lower than

A_ts1_1 and confirms the need of a relaxed six-membered ring

system. The rate limiting step in the mechanism is to attain

A_ts1_1, for which the barrier is 40.5 kcal mol�1. Allowing for

extra water molecules to participate in the reaction, the barrier

for the associative mechanism of [MeOPO3H]� hydrolysis is

about 1.6 kcal mol�1 lower than for [MeNHPO3H]�. Our

results at MP2/6-311**G++//B3LYP/DGDZVP level of

theory and previously reported calculations at different levels

of theory on [MeNHPO3H]� hydrolysis differ only by about

1–2 kcal mol�1 (Table 4) which validates the methods used in

this work.

A second mechanism is the interchange mechanism33 (Ia).

The interchange mechanism as presented here is a single step

mechanism with cis-attack and is to our knowledge not

reported for [MeOPO3H]�. Our own calculations and scans

of the free energy surface in the literature11 suggest that this

Table 1 Enthalpy of phosphate hydrolysis in kcal mol�1. Thereactions are presented in Fig. 1

G3B3 G3MP2 G3MP2

XQNH �7.7 �7.7 R1QOH, R2QOH 1.8XQO 1.7 1.6 R1QOH, R2QO� �58.7XQS �6.4 –5.9 R1QO�, R2QO� �127.9

Fig. 2 Geometries of the reactants used in the G2MP2 calculations

on L-Asp-phosphoramidate [MeNHPO3H]� hydrolysis.

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Table 2 Enthalpy of phosphate hydrolysis in kcal mol�1. Basis set for HF,B3LYP,MP2 = 6-31++G**

G3B3 HF27 B3LYP27 MP227

CH4P3O10� + H2O - CH4P2O7

� + H3PO4 14.5 13.7 14.5 12.6CH4P3O10

2� + H2O - CH4P2O7� + H2PO4

� �47.9 �48.5 �48.8 �48.5CH4P3O10

3� + H2O - CH4P2O72� + H2PO4

� �96.4 �168.4 �109.3 �86.0

Fig. 3 Stationary points on the pathways for the associative and interchange mechanism of [MeNHPO3H]� hydrolysis.

Table 3 Relative energies for stationary point in [MeNHPO3H]-hydrolysis (in kcal mol�1)

B2LYP/DGDZVP MP2/6-311++G** //B3LYP/DGDZVP

DEg DGg DGsol DGaq DEg DGg DGsol DGaq

AssociativeReact_1 0.0 0.0 �69.3 0.0 0.0 0.0 �72.4 0.0React_2 0.0 0.0 �67.5 0.0 0.0 0.0 �70.5 0.0A_ts1_1 42.7 44.7 �71.2 42.8 40.5 42.5 �74.3 40.5A_ts1_2 42.3 45.4 �68.7 44.2 40.3 43.4 �71.1 42.8A_int_1 34.6 36.8 �69.1 36.9 35.1 37.3 �71.3 38.4A_ts2_1 44.6 46.6 �67.9 48.0 43.2 45.2 71.3 47.7A_ts2_2 38.8 42.6 �68.8 41.2 37.5 41.3 71.5 40.3

InterchangeIa_ts1_1 53.4 55.3 �84.0 40.5 54.7 56.5 �89.2 39.6

Tautomeric associative and dissociativeTA/TD_ts1a_1 28.0 28.3 �69.7 27.9 27.5 27.8 �73.7 26.7TA/TD_ts1b_1 14.9 16.8 �75.6 10.4 14.5 16.5 �80.2 8.7TA/TD_int_1 7.8 7.4 �77.5 �0.7 12.9 12.5 �81.7 3.5TA_ts2_1 41.1 40.6 �74.9 35.1 42.6 42.6 �77.3 37.4TD_ts2_1 11.7 11.1 �62.6 17.9 16.7 16.1 �65.6 23.2TD_int2_1 6.9 3.8 �56.7 16.5 13.4 10.3 �60.9 22.1TD_ts3_1 31.4 20.0 �63.3 36.1 35.3 33.9 �66.5 40.0TA/TD_ts3_2 25.4 24.9 �67.1 25.3 28.9 28.5 �70.2 28.8

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mechanism does not exist for [MeOPO3H]�. We report it here

for [MeNHPO3H]�. This is a single step mechanism where

P–N bond breaking and P–O1 bond formation happen at the

same time (Fig. 3). Dissociation of the P–N bond is assisted by

a proton transfer from water to N. The P–N bond distance is

1.98 A, the P–O1 distance 2.13 A. The H1–N bond (1.06 A)

is almost completely formed in this step. The P–O2 bond is

enlarged from 1.67 A in the reactant complex to 1.77 A in this

transition state. The O2–P–O3, O2–P–N, O2–P–O4 and

O1–P–O2 angles are, respectively 97, 83, 100 and 1531. The

geometry is not a trigonal bipyramid as in the associative

mechanism, where the tetragonal phosphorus is approached

perpendicular to the plane formed by O3–O4–P, here the

approach is not perpendicular but displaced towards the

nitrogen vertex. This is caused by the hydrogen bond formed

between O1 and N–H. The DGg for this interchange mechanism

is 14.0 kcal mol�1 higher than for the associative mechanism.

But Ia_ts1_1 is stabilized by DGsol by 16.9 kcal mol�1 due to

the high charge separation in this TS. As a result the barrier is

0.9 kcal mol�1 lower than for the associative mechanism. In a

2D plot (see Fig. 4) it is illustrated that this mechanism is

situated between the associative and the dissociative pathway

but closer to the associative.

The transition states and intermediates for the third

mechanism are shown in Fig. 5. This is called a tautomeric

associative mechanism (TA), since this corresponds to

prototropic formation of a zwitterion followed by an attack

of water to the face opposite to the leaving group with

inversion of configuration. The first step is a proton transfer

from O2 to N with a partial rupture of the P–N bond. The

P–N bond distance enlarges to 1.95 A. H2 is only partially

transferred to N. This TS with direct proton transfer is a

very strained four-membered ring system with a barrier of

26.7 kcal mol�1 (Table 3). With a water molecule mediating

this proton transfer, the barrier is reduced by about

18 kcal mol�1 resulting in a barrier of only 8.7 kcal mol�1 in

TA_ts1b_1. TA_ts1b_1 is a concerted proton transfer, H2 is

transferred to O1 and at the same time H3 is transferred to N.

In this transition state the O2–H2 distance is 1.09 A and H3–N

distance 1.13 A indicating that the protons are mostly situated

on O2 and N. The P–N distance is 1.93 A here, which is

shorter than in A_ts2_2, this illustrates the importance of P–N

distance for the geometry of the six-membered ring. Compared

to [MeOPO3H]� (Table 4) TA_ts1b_1 is about 16 kcal mol�1

lower. This is consistent with the thermochemical parameters

for P–N which point out that a P–O bond is stronger than a

P–N bond. Further breaking of the P–N bond results in a

metaphosphate, P–N bond is only partially broken and P–N

distance is 2.15 A. DGg is 12.5 kcal mol�1 for TA_int_1, this

intermediate is largely stabilized by the solvent resulting in

DGaq of 3.5 kcal mol�1. The next rate-determining step is an

attack of water to this metaphosphate. We should note here

that methylamine is not completely dissociated from the

metaphosphate at this point. In TA_ts2_1 the metaphosphate

is attacked by water and the P–N distance is enlarged to

2.41 A, this is larger than in the associative and interchange

mechanism, but not a complete dissociation. The O1–P dis-

tance is 2.21 A which is similar to the first step in the

associative mechanism. The proton H1 is transferred to one

of the oxygen atoms of the metaphosphate. The role of a water

dimer in this step is discussed with the tautomeric dissociative

mechanism, since addition of a water dimer on TA_int1_2

results in TA/TD_ts3_2. The barriers for the tautomeric

Table 4 Relative energies of [MeOPO3H]� hydrolysis (in kcal mol�1)

B3LYP/DGDZVPaMP2/6-311++G**//B3LYP/DGDZVPa

B3LYP/cc-PVTZ+//B3LYP/6-31+G(d,p)b

CCSD(T)/(G2X)//B3LYP/6-31+G(d)c

DGaq DGaq DGaq DGaq

AssociativeReact 0.0A_ts1_1 40.9 38.6 36.1 37.5A_ts1_2 39.9 39.1A_ts2_1 40.6 39.1 37.6 38.6A_ts2_2 41.2 38.9

DissociativeTD_ts1a_1 38.8 36.5 35.9 33.4TD_ts1b_1 27.4 24.3 24.0 23.8TD_ts3_1 39.5 38.6 32.3 33.1TD_ts3_2 26.3 24.7 23.6

a This work. b Wang et al.10 c Hu et al.with DE[CCSD(T)/(G2X)]= DE[MP2/6-311+G(2df,2p)]� DE[MP2/6-31+G*]+ DE[CCSD(T)/6-31+G*].30

Fig. 4 Bond distances in A of P–O bond broken in function of

P–N bond formed along the different mechanisms. - � - �= associative,

– = interchange, ���� = tautomeric associative, —– = tautomeric

dissociative.

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associative mechanism are lower than for the associative and

interchange mechanism by at least 5 kcal mol�1.

Finally, as a fourth mechanism we have the tautomeric

dissociative (TD) mechanism. Here an internal proton transfer

is followed by a complete dissociation of methylamine before

water attacks the metaphosphate. First transition states and

intermediates are the same as in the tautomeric associative

mechanism. In contrast to the TA mechanism an elongation of

the P–N bond precedes the attack of a water molecule as

shown in TD_ts2_1 of Fig. 5. TD_ts2_1 is a total rupture of

the P–N bond (2.98 A) and formation of a true meta-

phosphate. Here we see a true dissociative mechanism, meta-

phosphate is now ready to be attacked by a water molecule.

TD_ts3_1 shows the attack of a water monomer on the

metaphosphate, here the P–N bond is completely dissociated

and the P–O bond of the incoming water is shorter than in

TA_ts3_1. The resulting barrier is 36.1 kcal mol�1. Here again

we see the importance of a water dimer in the mechanism. An

attack of a water dimer on metaphosphate results in a reduced

barrier. In TD_ts3_2 the barrier is reduced by 11.2 kcal mol�1.

Our results on the tautomeric dissociative mechanism of

[MeOPO3H]� hydrolysis are close to previous results

(Table 4), except for TD_ts3_1 with a difference of about

6 kcal mol�1, most probable due to the different solvent

treatment. These results indicate that for [MeNHPO3H]� the

TA or TD mechanism is preferred and for [MeOPO3H]� the

TD mechanism, certainly when the effect of water dimers on

the barriers is taken in account. The TA mechanism for

[MeNHPO3H]� hydrolysis is in good agreement with the

mechanism proposed by Chanley et al.34 based on their

experimental results. They see a complete quenching of the

hydrolysis at pH higher than 10, due to deprotonation of

[MeNHPO3H]�. For [MeOPO3H]� TD_ts1_1 has the highest

barrier, while for [MeNHPO3H]� this is TD_ts3_2. The largest

difference in barrier for both molecules is found for TD_ts1_1,

indicating that the bond breaking occurs much easier for

[MeNHPO3H]�. Overall we see for [MeNHPO3H]� that the

more we shift to a dissociative mechanism the lower the

barriers get.

Two papers exist where free energy surfaces of phosphate

hydrolysis are plotted as a function of the P–O bond formed

and broken.11,35 In our work no full scans were performed but

the formation of the P–O bond was plotted as a function of the

breaking of the P–N bond for the different stationary points

(Fig. 4). For clarity, only the mechanisms involving a water

monomer are plotted. This gives an overview of the nature of

the pathway. The associative mechanism passes via the lower

left of the graph, the tautomeric dissociative via the upper

right, those are the two extreme pathways. Very close to the

associative pathway we find the interchange pathway. We also

see that the TD and TA mechanisms coincide in the

initial phase.

C Hydrolysis reaction with aspartic acid as leaving groups

As a first model for L-Asp-dAMP3,4 hydrolysis we opted for

N-(methoxyphosphinato)aspartate (Fig. 6). Similar to the

model used by Bojin et al.36 we added a methyl group to

one of the oxygens of the phosphate modeling the ribose and

the base. The choice of this model implies that the TA and TD

mechanism are excluded since the proton responsible for the

internal proton transfer is replaced by a methyl. Hence only

the A and Ia mechanisms are possible. The carboxyl functions

were deprotonated since this is the expected state at

physiological conditions. The pKa of the side chain carboxyl

is 3.9 and that of the backbone carbonyl 2.1.37 The reactant

complex is shown in Fig. 6. There is a hydrogen bond between

the methyl group on phosphoramidate and one of the carboxyl

groups. The P–N bond length is 0.02 A smaller than in the

reactant complex of [MeNHPO3H]�. The attacking water

molecule forms hydrogen bonds with the phosphoramidate

and one of the carboxyls of aspartic acid.

For the associative mechanism the high charge of the

compound made optimization at B3LYP/DGDZVP level

impossible for A_ts1_1 (Fig. 6). This structure and A_int_1

were optimized at HF/3-21G* level and then the single point

energy was calculated at B3LYP/DGDZVP level. All other

structures were optimized at B3LYP/DGDZVP level. This

Fig. 5 Stationary points on the pathways of the tautomeric associative and tautomeric dissociative mechanisms of [MeNHPO3H]� hydrolysis.

Only metaphosphate and attacking water molecules are shown for TA/TD_ts3_2.

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makes it difficult to compare the geometry of A_ts1_1 to the

other calculated reactions. No reactions were calculated using

extra water molecules here due to the uncertainty caused by

the high charge of the system. Qualitatively this transition

state is similar for [MeNHPO3H]� and [MeOPO3H]�. DEg is

69.8 kcal mol�1 using B3LYP, a single point MP2 calculation

reduces the energy to 64.4 kcal mol�1. It should be stressed

that this is just an estimate, we expect the true barrier to be

lower. The solvation correction for this transition state is very

large, this is caused by the large charge separation in this

transition state, due to the proton transfer from water to

phosphoramidate leaving a negatively charged OH�. DGaq at

B3LYP level is 57.2 kcal mol�1 which is almost 15 kcal mol�1

larger than for [MeNHPO3H]�. Similar to [MeOPO3H]�10,30

and [MeNHPO3H]� this reaction proceeds through a trigonal

bipyramidal intermediate. Before A_ts2_1 can occur, the H1

must rotate towards N, the transition state for this conforma-

tional change is not shown here. In A_ts2_1 a H1 is transferred

from O3 to N, at the same time the P–N bond is partially

broken. Again, taking into account solvent stabilization, the

energy is reduced from 74.6 to 59.4 kcal mol�1 at B3LYP level

and from 69.6 to 53.5 kcal mol�1 at MP2 level. The O1–P

distance is 1.79 A and the P–N distance 2.07 A, in

[MeNHPO3H]� those were, respectively 1.74 and 2.18 A for

[MeNHPO3H]�. Also the barrier here is 12.7 kcal mol�1

higher. Overall the higher charge has a large destabilizing

effect on the associative mechanism.

For the interchange mechanism Ia_ts1_1 (given in Fig. 6) the

transfer of H1 to N and the formation of the P–O1 bond occur

at the same time. The P–O1 distance is 2.23 A and the P–N

distance 1.93 A, so the formation of the P–O1 bond and

breaking of the P–N bond happen at the same time. The

geometry of this complex has some similarities to the

associative mechanism where methanol would be the leaving

group. The angles O2–P–N, O2–P–O4, O2–P–O3 and

O1–P–O2 are, respectively, 91, 97, 100 and 163. The O2–P

bond is 1.75 A and the O1–P distance 2.23 A, which corres-

ponds to a highly distorted trigonal bipyramid. However, as

confirmed by intrinsic reaction coordinate (IRC) calculations

aspartic acid is the leaving group in this transition state and

not methanol. Again the energy is reduced upon solvation

from DEvac = 54.3 kcal mol�1 to DGaq = 46.2 kcal mol�1.

The barrier for this mechanism is considerably lower than for

the associative mechanism. Our results at B3LYP level suggest

a reduction by 11.7 kcal mol�1, at MP2 level this is reduced to

7 kcal mol�1. Compared to the interchange mechanism in

[MeNHPO3H]� the barrier is 6.8 kcal mol�1 higher. In

general, we find here that an interchange mechanism is

preferred and that the barrier in this highly charged compound

is higher than in [MeNHPO3H]�.

Compared to triphosphate reactions reported in recent

papers10,11 barriers reported here for L-Asp-phosphoramidate

are much higher. Comparing the associative mechanism, Evac

for A_ts1_1 is about 22 kcal mol�1 higher and A_ts_2 is about

27 kcal mol�1 higher than the values reported for the

similar mechanisms at B3LYP/6-31+G(d,p) level of theory

in triphosphate.10 In this paper10 solvent stabilization for

triphosphate is similar to the one reported here for N-(methoxy-

phosphinato)aspartate. Although it is difficult to draw

conclusions on this highly charged species the calculations

reported here suggest that the hydrolysis reaction of

L-Asp-phosphoramidate has a higher barrier than tri-

phosphate hydrolysis and that the interchange mechanism is

preferred. The high values found for the barriers seem to

Fig. 6 Stationary points on the pathways of the associative and interchange mechanism of hydrolysis reaction with aspartic acid as leaving group

(charge = �3).

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emphasise that these reactions are possibly unfeasible in

the absence of a cationic catalyst. For this reason we

introduced Mg2+.

D Hydrolysis reaction using Mg2+

with malic and aspartic

acid as leaving groups

Many enzymes involved in reactions with triphosphate have

Mg2+ in their active site. We are especially interested in

polymerases.38–40 Adelfinskaya et al.3 built a model of the

active site of HIV-RT with L-Asp-dAMP using a known

crystal in DOC structure with a dideoxynucleotide in the

active site.41 We added Mg2+ to the different minima found

for N-(methoxyphosphinato)aspartate and compared to the

structures taken from the model of Adelfinskaya et al.3 and

minimized at the same level of theory. It was found that the

structure from the model of Adelfinskaya et al.3 had the lowest

energy. We continued to calculate hydrolysis reaction with this

structure.

The reactant structure is shown in Fig. 7. The two carboxyl

groups of aspartic acid, O4 and N, are coordinating Mg2+; to

obtain a six-coordinated Mg2+ and complete the solvation

shell38,39,41 of Mg2+ we added two water molecules similarly

to Klahn et al.11 The result is a distorted octahedral coordination

for Mg2+. The large charge (�3) in the previous model is

reduced to �1 by adding Mg2+. The attacking water molecule

is hydrogen bonded to the phosphate of the reactant complex

this time. The P–N bond in the reactant complex is 0.03 A

longer here than in Fig. 6 for the structure without Mg2+.

Relative energies of hydrolysis reaction are displayed in

Table 5.

A_ts1_1 is similar again to the previously calculated

reactions. The P–O1 distance is 2.21 A here and the P–N

distance 1.76 A. Comparison to the previous section where the

same molecule was calculated without Mg2+ must be done

with care since the geometry in the previous section was

calculated at a lower level. We see large differences in P–N

and P–O1 bond lengths in Fig. 7 but it is impossible to draw

conclusions from this. Again we compared this reaction to a

reaction with a water dimer. In line with the calculations on

[MeNHPO3H]� this is not lowering the energy. Attack of

OH� results again in the typical trigonal bipyramidal inter-

mediate A_int_1. After rotation of H1 towards N we arrive at

A_ts2_1, here H1 is transferred to N and the P–N bond is

broken. The P–N distance here is 2.34 A, the P–O1 distance

1.67 A. Compared to the reaction without Mg2+ the P–N

bond is larger and P–O1 smaller, indicating that the P–O1

bond is already largely formed and the P–N bond more

dissociated. The Mg2+ is weakening the P–N bond and

stabilizing the negatively charged nitrogen in this TS. The

longer P–N bond in this TS suggests that an extra water

molecule will not catalyze this reaction further. This is

confirmed in Table 6, it is even destabilizing A_ts2_1 by

8.7 kcal mol�1 at B3LYP level and by 13 kcal mol�1 at

MP2 level, this is in large contrast to [MeNHPO3H]� where

the barrier is reduced by 6.77 kcal mol�1 by using a six-

membered ring. A_ts1_1 and A_ts2_1 are very close in energy

at B3LYP level, the difference is larger at MP2 level, confirming

that A_ts2_1 has the highest energy, as was also the case for

the same system without Mg2+. The barrier for the rate

limiting step is 38.8 kcal mol�1 at B3LYP level and

37.4 kcal mol�1 at MP2 level. Comparing with the system

without Mg2+ the reduction of the barrier is spectacular,

by 19.8 kcal mol�1 at B3LYP level and by 12.4 kcal mol�1

at MP2 level. Still the barrier is about 8 kcal mol�1 higher than

for a similar model with diphosphate.11

The associative mechanism was also calculated for a similar

system with malic acid instead of aspartic acid as leaving

Fig. 7 Stationary points on the pathways of the associative and interchange mechanism of hydrolysis reaction using Mg2+ with aspartic acid as

leaving group (charge of the hydrolysed molecule = �3, charge of total system = �1).

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group. This is largely the same system except that the leaving

group is coupled with a P–O bond rather than P–N. There is

only one TS along this pathway, the lowest barrier here

is using two water molecules (A_ts1_2), for this TS the barrier is

36.2 kcal mol�1. The A_ts2 transition state doesn’t exist here:

upon rotation of H1 the proton is directly transferred to the

oxygen of the leaving group. The difference in barrier between

the N coupled systems is 1 kcal mol�1 at B3LYP level and

1.1 kcal mol�1 at MP2 level. As also seen for [MeOPO3H]�

and [MeNHPO3H]�, for the associative mechanism O-coupled

leaving groups have lower barriers than N-coupled leaving

groups. The barrier here is closer to the barrier of

32 kcal mol�1 estimated for diphosphate by Klahn et al.11

In Fig. 7 Ia_ts1_1 for the reaction with aspartic acid is

shown. This is the transition state for a single step mechanism.

P–N distance is 1.94 A and P–O1 distance is 2.17 A. Both are

shorter than in the system without Mg2+. The geometry

around the phosphorus is even more distorted from a trigonal

bipyramid: O2–P–O4, O3–P–O4, N–P–O4 and O1–P–O4

angles are, respectively 108.68, 100.03, 93.7 and 160.2. For

the proton transfer from O1 to nitrogen to occur an inversion

at the nitrogen is necessary. This explains the higher barrier for

this mechanism compared to the mechanism without Mg2+.

Using B3LYP the barrier with Mg2+ is about 3 kcal mol�1

higher, with MP2 it is 5 kcal mol�1 higher than in the absence

of Mg2+. The barrier is around 12.1 kcal mol�1 higher than

the equivalent barrier for [MeNHPO3H]�. In the presence of a

single Mg2+, the barrier for the interchange mechanisms is

considerably higher than that for the associative mechanism,

by 11.3 kcal mol�1 at B3LYP level and 12.9 kcal mol�1 at

MP2 level. We conclude that the reaction with Mg2+ occurs

preferentially by an associative mechanism. This is also the

mechanism proposed by Bojin et al.36 and Alberts et al.40 for

the polymerization reaction of polymerase b.

E Hydrolysis reaction using two Mg2+

with aspartic

and malic acids as leaving groups

As a final model we involve a second Mg2+ ion in the reaction

(Fig. 8). This second metal ion, the catalytic Mg2+ is an

electrophilic catalyst that stabilizes the charge of the hydroxide

ion attacking the phosphorus. Polymerases often make use of

those two Mg2+ ions.39,40,42 The relative energies for

hydrolysis reaction using Mg2+ with malic acid as leaving

group are given in Table 7. We again started from the model

built in the work of Adelfinskaya et al.3 We kept the groups

coordinating Mg2+ and added the attacking water molecule.

The reacting molecules are the same as before, an overview is

given in Fig. 9. The catalytic Mg2+cat is coordinated by five

ligands: an oxygen of asp110, of asp185, of asp186, of

phosphate and of water. Different from some previous models

we did not include an extra water molecule to obtain

six-coordinated Mg2+. The aspartic acid coordinating

Mg2+asp is linked to six ligands: oxygen of val111, of asp110,

of asp185, of the carboxyl groups of aspartic acid and of

phosphate. Different from the previous model but similar to

the model of Adelfinskaya et al.3 the N is not in the first

coordination sphere of the Mg2+. This model is similar to

models proposed for DNA-polymerase b36,43 or HIV-RT.38 In

Table 8 we reported the distances for the coordination of

Mg2+ and compared them to the X-ray structure41 and the

model by Adelfinskaya et al.3 All structures in this section

are optimized at B3LYP/6-31G level. Remarkable is the

Mgcat-OD1(Asp186), Asp186 migrates closer to Mgcat by

1.84 A, this was also the case in the simulations of

Adelfinskaya et al.3 and in some models of Rungrotmongkol

et al.39 Overall there are no large differences in distance

between the averages of simulations of Adelfinskaya et al.3

and the active site optimization done here.

Table 5 Relative energies of hydrolysis reaction with aspartic acid as leaving group (charge = �3) (in kcal mol�1)

B3LYP/DGDZVP MP2/6-311++G** //B3LYP/DGDZVP

DEg DGg DGsol DGaq DEg DGg DGsol DGaq

React 0.0 0.0 �368.2 0.0 0.0 0.0 �372.2 0.0A_ts1_1a 69.8 70.8 �385.5 57.2 64.4 69.4 �387.6 53.2A_int_1a 64.4 69.5 �385.8 51.9 58.2 63.3 �387.7 48.0A_ts2_1 71.4 74.6 �381.0 59.4 65.8 69.6 �384.7 53.5Ia_ts1_1 54.6 55.5 �375.8 47.7 54.3 55.2 �380.6 46.2

a This structure was only optimized at HF/3-21G* level, reported energies are single point energies.

Table 6 Relative energies for hydrolysis reaction using Mg2+ with aspartic acid as leaving group (charge of the hydrolysed molecule = �3,charge of total system = �1) (in kcal mol�1)

B3LYP/DGDZVP MP2/6-311++G** //B3LYP/DGDZVP

DEg DGg DGsol DGaq DEg DGg DGsol DGaq

React_1 0.0 0.0 �65.1 0.0 0.0 0.0 �72.6 0.0React_2 0.0 0.0 �67.1 0.0 0.0 0.0 �71.7 0.0A_ts1_1 35.1 37.4 �64.1 38.4 33.6 35.9 �71.2 37.4A_ts1_2 35.6 38.5 �65.9 39.7 33.9 36.8 �71.2 38.8A_int_1 24.4 26.8 �65.0 26.8 25.0 27.3 �71.7 28.2A_ts2_1 36.4 39.6 �64.4 39.6 36.5 38.9 �70.5 41.1A_ts2_2 45.0 46.6 �65.5 48.3 44.3 46.0 �65.5 54.2Ia_ts1_1 60.6 61.6 �76.6 50.9 63.4 64.5 �86.1 51.7

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Only the associative mechanism was calculated here, several

other studies on similar systems point out that an associative

mechanism is preferred, 36,40 moreover the geometry of the

HIV-RT active site makes an interchange mechanism impossible.

As before, the highest barrier is the deprotonation step with a

concerted attack of the hydroxide ion to phosphorus in A_ts1_1.

The Mg2+ O1 distance decreases by 0.07 A to

2.00 A. We see here that involving the Mg2+cat lowers this barrier

by 8.9 kcal mol�1. The relative energies are given in Table 9. Our

model is very similar to themodel of Bojin et al.36 for polymerase b.An important difference is that in their model the phosphorus

is attacked by methanol and the leaving group is pyrophosphate.

The value they computed for the first intermediate is

28 kcal mol�1 at B3LYP/6-31G(d,p) level compared to our value

for aspartate of 29.5 kcal mol�1 on B3LYP/DGDZVP level. This

indicates that the barrier for triphosphate hydrolysis will be

slightly lower than for aspartic acid. No transition states were

optimized in the work of Bojin et al., they estimated that the

barrier was 15 kcal mol�1 higher than the first intermediate,

which is much too high compared to the experimental value of

16 kcal mol�1 in polymerase b. Finally if we compare to the

reaction with malic acid as a leaving group (see Table 10) we

observe that the barrier is 1.5 kcal mol�1 lower, indicating that

malic acid might be a better alternative for pyrophosphate. The

reaction proceeds similarly to previous reaction with one Mg2+.

A_ts2_1 is higher by 2.3 kcal mol�1, which might be expected

since N is not linked to Mg2+ as in the previous model.

IV. Conclusion

Recalculation of triphosphate hydrolysis reactions at higher level

of theory and comparison to hydrolysis of phosphoramidates

with aspartic acid as leaving group confirms that replacing

Fig. 8 Stationary points on the pathways of the associative mechanism of hydrolysis reaction using two Mg2+ with aspartic acid as leaving group

(charge of the hydrolysed molecule = �3, charge of total system = �2).

Table 7 Relative energies for hydrolysis reaction using Mg2+ with malic acid as leaving group (charge of the hydrolysed molecule = �3, chargeof total system = �1) (in kcal mol�1)

B3LYP/DGDZVP MP2/6-311++G** //B3LYP/DGDZVP

DEg DGg DGsol DGaq DEg DGg DGsol DGaq

React_1 0.0 0.0 �66.3 0.0 0.0 0.0 �73.6 0.0React_2 0.0 0.0 �65.9 0.0 0.0 0.0 �71.7 0.0A_ts1_1 34.0 36.5 �65.4 37.4 32.2 34.7 �72.0 36.3A_ts1_2 34.4 37.2 �66.5 37.2 32.0 34.9 �72.6 35.5Ia_ts1_1 43.9 45.9 �67.5 45.7 46.2 48.2 �75.3 47.6

Fig. 9 Model of the active site of HIV-RT with N-(methoxy-

phosphinato)aspartate.

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pyrophosphate by aspartic acid results in higher hydrolysis

enthalpies. Also the trend when increasing the charge is similar.

This indicates that from a point of view of thermodynamics

aspartic acid can serve as a good replacement of pyrophosphate

where the P–N bond in phosphoramidate is even less stable than

the high energy P–O bond in triphosphate.

From calculations on the simplest systems [MeNHPO3H]�

and [MeOPO3H]� we can draw three important conclusions.

The first is that the use of a water dimer plays a crucial role in

lowering the barrier for the tautomeric dissociative mechanism

and tautomeric associative mechanism, while it does not

reduce the barrier significantly for the other mechanisms.

The second conclusion concerns the hydrolysis mechanisms,

we see that two extra mechanisms are possible when we

compare [MeNHPO3H]� to [MeOPO3H]�, the interchange

mechanism and the tautomeric associative mechanism. We

also see a strong preference for a tautomeric mechanism where

a zwitterionic intermediate is formed for [MeNHPO3H]�

hydrolysis. A third conclusion arises from comparing

[MeNHPO3H]� to [MeOPO3H]� for both a tautomeric

mechanism if preferred. Comparing the associative mechanisms

the barriers are 40.5 and 38.6 kcal mol�1 for [MeNHPO3H]�

and [MeOPO3H]�, respectively. The pathway to achieving

a metaphosphate is much lower for [MeNHPO3H]� than

for [MeOPO3H]� this might indicate that phosphoramidates

are better substrates for enzymes using a dissociative

mechanism.44 While in enzymes using an associative

mechanism (which is expected for HIV-RT with normal

nucleotides) the barrier for pyrophosphate is lower than for

L-Asp-NMP. This is also seen in the experiments by

Adelfinskaya et al.3

Subsequently aspartic acid was compared as a leaving group

to previous work on triphosphate. Barriers for this highly

charged compound were much higher than for

[MeNHPO3H]� and for triphosphate. The choice of the model

excluded the possibility of a dissociative mechanism and

because of the high charge it was found difficult to optimize

TS. Here the interchange mechanism is preferred but the

barrier is much higher than for triphosphate, emphasising

the need for cationic agents.

Table 8 Active site distances for crystal structure,41 simulations with the amber99 force field of Adelfinskaya et al.3 and QM optimization(B3LYP/6-31G), in A

X-Ray41 Adelfinskaya et al.3 QM optimization

Mgcat–H2O 2.07Mgcat–O30(primer terminus) 2.09Mgcat–OD2(D110) 2.68 1.90 2.01Mgcat–OD1(D185) 2.05 1.88 2.04Mgcat–OD1(D186) 3.87 1.83 2.03Mgcat–O4 (P) 3.03 2.38 2.09Mgasp–OD1(D110) 2.13 1.92 2.12Mgasp–O(V111) 2.27 2.06 2.18Mgasp–OD1(D185) 2.36 1.88 2.06Mgasp–O4 (P) 2.21 2.07 2.13Mgasp–OD2 (Asp) 1.89 2.04Mgasp–OD3 (Asp) 1.95 2.12H2O–P 3.25O30(primer terminus)-P 3.09Mgcat–Mgasp 3.57 3.70 3.53

Table 9 Relative energies for hydrolysis reaction using two Mg2+ with aspartic acid as leaving group (in kcal mol�1), (charge of the hydrolysedmolecule =�3, charge of total system = �2)

B3LYP/6-31G B3LYP/DGDZVP //B3LYP/6�31G

DEg DGg DGsol DGaq DEg DGg DGsol DGaq

React 0.0 0.0 �161.1 0.0 0.0 0.0 �152.4 0.0A_ts1_1 21.4 22.0 �161.0 22.2 30.0 30.6 �153.4 29.5A_int_1 20.6 21.0 �161.4 20.7 30.1 30.5 �156.8 29.1A_int_1 22.8 23.6 �164.1 20.6 31.4 32.2 �156.0 28.5A_ts2_1 32.1 32.0 �163.1 31.0 43.7 44.6 �155.0 42.0

Table 10 Relative energies for hydrolysis reaction using two Mg2+ with malic acid as leaving group (charge of the hydrolysed molecule = �3,charge of total system = �2), (in kcal mol�1)

B3LYP/6-31G B3LYP/DGDZVP //B3LYP/6-31G

DEg DGg DGsol DGaq DEg DGg DGsol DGaq

React 0.0 0.0 �159.8 0.0 0.0 0.0 �150.8 0.0A_ts1_1 21.7 22.2 �160.5 21.4 29.0 29.5 �152.4 28.0A_int_1 19.0 19.1 �162.9 16.0 28.7 28.8 �154.5 25.1A_int_1 16.2 16.8 �162.5 14.0 27.6 28.2 �154.8 24.2A_ts2_1 29.0 29.6 �163.1 28.2 43.3 43.8 �153.6 41.1

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For this reason we introduced Mg2+. Barriers for the

associative mechanism were spectacularly reduced compared

to the model without Mg2+ and slightly compared to

[MeNHPO3H]�. Here barriers are still higher than the

estimate for triphosphate with one Mg2+ of Klahn et al.11

and can be reduced by about 1 kcal mol�1 using malic acid as

leaving group.

Finally inclusion of two Mg2+ ions in the reaction using an

active site model reduces the barrier to 29.52 kcal mol�1.

Compared to the system with one Mg2+ this is a reduction

of 8.9 kcal mol�1. This demonstrates the crucial role of the

second Mg2+ ion. The barrier for N-(methoxyphosphinato)-

aspartate is always higher in energy than for triphosphate.

It can be reduced by the use of malic acid. Comparison of our

intermediates to the intermediates in the model of Bojin et al.36

suggests that with malic acid as a leaving group the barrier is

similar to triphosphate while with aspartic acid the barrier is

higher. The present QM results also leave little doubt that the

proper barrier for hydrolysis is much higher than the actual

barrier in the enzymatic reaction. We suspect that the protein

environment plays a crucial role in promoting fast proton

transfer. A further detailed hybrid QM/MM study will be

needed to elucidate the catalytic role of the protein. We can

conclude from comparison of this study with previous results

in the literature on triphosphate hydrolysis that barriers are

comparable but slightly higher and that malic acid might be an

improvement over aspartic acid as a leaving group.

Acknowledgements

This work has been supported by the Fund for Scientific

Research-Flanders (FWO). Janne Michielssens is acknowledged

for carefully reading the paper.

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