can arsenates replace phosphates in natural biochemical...
TRANSCRIPT
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Can Arsenates Replace Phosphates in Natural
Biochemical Processes? A Computational Study
Jissy, A. K.1, Ayan Datta
1,2*
1School of Chemistry, Indian Institute of Science Education and Research Thiruvananthapuram,
CET Campus, Thiruvananthapuram-695016, Kerala (India).
2Department of Spectroscopy, Indian Association for the Cultivation of Science, Jadavpur – 700032,
West Bengal (India).
ABSTRACT: A bacterial strain, GFAJ-1 was recently proposed to be substituting arsenic for
phosphorus to sustain its growth. We have performed theoretical calculations for analyzing this
controversial hypothesis by examining the addition of phosphate to ribose and glucose. Dispersion
corrected Density Functional Theory (DFT) calculations in small molecules and QM/MM
calculations on clusters derived from crystal structure are performed on structures involved in
phosphorylation, considering both phosphates and arsenates. The exothermicity as well as the
activation barriers for phosphate and arsenate transfer were examined. Quantum mechanical studies
reveal that the relative stability of the products decrease marginally with successive substitution of P
with As. However, simultaneously, the transition state barriers decrease with P replacement. This
indicates that kinetically, addition of As is more facile. A glucokinase crystal structure was chosen
to construct a model system for QM/MM calculations. Free energy of the reaction (∆G) reduces by
less than 2.0 kcal/mol and the activation barrier (∆G‡) decreases by ~ 1 kcal/mol on arsenic
incorporation. Thus, both DFT and QM/MM calculations show that arsenic can readily substitute
phosphorus in key biomolecules. Secondary kinetic isotope effects for phosphorylation mechanism
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obtained by QM/MM calculations are also reported. The secondary kinetic isotopic effect (SKIE)
for ATP and ATP (As) are calculated to be 5.81 and 4.73, respectively. A difference of ~ 1.0 in SKIE
suggests that it should be possible to experimentally determine the As – phosphorylation process.
KEYWORDS: Arsenate, phosphate, Density Functional Theory, QM/MM, kinases, kinetic isotopic
effect.
INTRODUCTION
In 2011, Simon et al reported the discovery of an unusual microbe strain GFAJ-1, which
could substitute arsenic for phosphorus and vary the elemental composition of its basic
biomolecules.1
The possibility of an alternative, arsenic-based life form, an element which is known
to be toxic, created much interest, but also raised concerns over the validity of this hypothesis.2-10
Arsenic as situated directly below phosphorus, shares many key chemical properties with the
element.11
Arsenate (HAsO4−2
) and phosphate (HPO4−2
) are abundant on earth, have nearly identical
pKa values,12
share similar thermochemical radii that differ by only 4%13
and forms analogous
esters. Irrespective of the similarities, phosphate is essential for life and arsenate is toxic. However,
there are several reasons for nature selecting phosphorus and not arsenic for synthesizing essential
biomolecules. Compared to phosphate esters, arsenate esters are highly unstable in water, and the
hydrolysis rates of arsenate esters are orders of magnitude greater than those of the corresponding
phosphates.14-18
Higher reactivity with water would also lead to problems for the stability of the
DNA/RNA containing arsenate-linked nucleic acids. In addition, a multitude of metabolic reactions
would have to substitute arsenate for phosphate. Thus the corresponding enzymes acting on
phosphate substrates would need to be mutated as the natural enzymes selected by nature are
generally inhibited by the arsenate substrates.
Mládek et al performed quantum chemical calculations to investigate the geometry and
electronic properties of the arsenate analogue of the DNA backbone.19
They concluded that arsenate
anions may behave as a perfect substitute for phosphate in DNA backbone. However, the
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incorporation of phosphates in biosystems begins from the phosphate transfer from ATP to different
molecules. Thus, by applying high-level quantum chemical calculations, we have analyzed the
incorporation of phosphates into the sugar moiety, which finally becomes a part of biomolecules. In
biosystems, D-ribose is one sugar which must be phosphorylated by the cell before it can be used.
Ribokinase is the enzyme which catalyzes this reaction by converting D-ribose to D-ribose-5-
phosphate. Once converted, D-ribose-5-phosphate is available for the synthesis of the nucleotides,
tryptophan and histidine, or can be used in the pentose phosphate pathway.20
ATP + D-ribose → ADP + D-ribose 5-phosphate
Thus, ATP and D-ribose are the two substrates leading to the formation of ADP and D-ribose 5-
phosphate. As D-ribose 5-phosphate is involved in the production of many important
biomolecules, it is instructive to study the phosphorylation of sugar with subsequent replacement of
phosphorus by its analogue, arsenic. We have thus, modeled our system based on this reaction
(Scheme 1). We have considered phosphoric acid, pyrophosphoric acid, diphosphono hydrogen
phosphate (DHP) and their arsenic analogues as substrates for addition of phosphate to ribose. The
interaction energies and activation barriers for phosphate and arsenate transfer are compared to
determine the feasibility of arsenic incorporation.
Combined quantum mechanical/molecular mechanical (QM/MM) methods have become an
important tool of choice for modeling enzyme catalyzed reactions and predicting the free energies
and activation barriers with high chemical accuracy.21
The substrate(s) and only the relevant
(conserved) residues in the active site are treated quantum mechanically. The rest of the protein is
described at the empirical molecular mechanics level. This makes it possible to treat large enzyme
systems as the computational cost is considerably lowered. We have constructed a model system
from a reported glucokinase crystal structure, for understanding phosphorylation of glucose and
ribose. QM/MM calculations are performed on the model system considering ATP and arsenylated
ATP as phosphate/arsenate donor.
One powerful kinetic tool for understanding enzyme reactions is the isotope effect.22
Isotope
4
effects provides information about the nature of the transition state in the rate determining step. If
the isotopic substitution at a site other than the bond-breaking site results in a change in the rate of
the reaction, it is known as secondary isotopic effect and can provide information about the role of
motion of functional groups/weakly bound molecules that are placed physically away from the bond
making/bond breaking site..23
The deutration of the three water molecules which are existing in the
glucokinase crystal structure and studied at the QM level in the QM/MM calculations are
considered. These three H2O/D2O molecules are in a combined motion in the transition state and are
analyzed to investigate the secondary kinetic isotopic effect of water on phosphorylation.
COMPUTATIONAL METHODS
All the quantum mechanical calculations were performed using the Truhlar’s M06-2X
version of DFT which has been remarkably successful in describing dispersion in a variety of
molecular systems.24,25
M06-2X functional is a highly parametrized metahybrid method which has
shown to be adequate for the study of intermediate range of electron-electron interactions involved
in enzyme catalysis.26
The triple-δ 6-311+G(d,p) basis set was employed.27
Additional frequency
calculations were performed to verify all positive vibrational normal modes for the ground state
structures and a single negative frequency for transition state (TS) structures. Solvent calculations
for all structures were also performed in water using the Polarizable Continuum model (PCM).28
In
PCM model, the solute is embedded in a cavity free of any solvent molecule, and the solvent
(water) is considered to be a continuum outside the cavity. The cavity is determined from the
molecular surface, as constructed from the scaled van der Waals radii of the constituent atoms.
The exothermicity of phosphory-/arseny-lation is evaluated as:
ΔG298K, 1atm
= GP298K, 1atm
– GR298K, 1atm
where ΔG is the free energy change, GP is the sum of the free energies of the optimized products
and GR is the sum of the free energies of the optimized reactants.
The crystal structure (PDB id: 3ID8) of human pancreatic glucokinase crystallized with
5
glucose and AMP-PNP was used to construct the model system for QM/MM computations.29
The
model reaction system was simplified to include sugar, ATP, Mg2+
, aspartic acid and lysine. The two
most important amino acid residues were included. All other residues were removed. The
unsatisfied valencies are satisfied by H-atoms. QM/MM calculations were performed using the two-
layer ONIOM method30
incorporated in Gaussian 09.31
QM/MM calculations on reactions in
enzymes with density functional theory has been successful in reproducing experimental results.32
The QM and the MM regions are calculated at B3LYP/6-31G(d) and AMBER, respectively.33
RESULTS and DISCUSSION
The interaction energy analyses of the reactions depicted in Scheme 1 are tabulated in Table 1.
In order to mimic ATP, methyl derivative of DHP was also considered, with the adenosine part
replaced by methyl group. For all the systems, gas phase calculations show that the addition of an
arsenate to ribose is energetically, slightly less favorable (~2.0 kcal/mol – 6.0 kcal/mol) as
compared to phosphate addition. The H-bond distances in phosphorylated- and arsenylated ribose
are 1.88 Å, 2.67 Å, 2.59 Å and 1.89 Å, 2.83 Å, 2.79 Å, respectively. Thus, the product formed by
phosphate transfer is more stable as compared to the arsenylated sugar. However, if we look at the
reactants, that is, DHP and its arsenic analogue with three arsenic atoms, the H-bond distances are
1.98 Å, 2.01 Å, 2.29 Å and 1.93 Å, 1.93 Å, 2.23 Å, respectively. The higher stability of products,
with the additional lower stability of reactants makes the reaction energetically more feasible for an
all-phosphorus substrate. The TS structures for phosphory-(arseny-)lation of ribose were also
determined. Figure 1 shows the TS structure for phosphate transfer from methyl-DHP to ribose.
The TS corresponds to the transfer of proton from sugar to phosphate. Subsequently the system
undergoes an internal proton transfer which leads to the rupture of the phosphoester bond and attack
of phosphate on the sugar hydroxyl group. Except for DHP, arsenic substitution results in a steep
decrease in the activation barriers. Phosphorylation of methyl-DHP involves crossing a barrier of
35.7 kcal/mol, whereas for 3As-methyl-DHP the barrier decreases by ~15.0 kcal/mol. We closely
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looked at the TS geometries for the reaction with methyl-DHP and its all-arsenic analogue. The H-
bond lengths in the TS for methyl-DHP are 1.87 Å, 1.97 Å, 2.9 Å and 2.2 Å which are longer then
the hydrogen bonds observed in the TS for arsenylated methyl-DHP (1.82 Å, 2.0 Å, 2.2 Å and 1.9
Å). Thus, the transition states are more stabilized by H-bonding interactions in 3As-methyl-DHP as
compared to methyl-DHP. Similarly, when pyrophosphoric acid acts as a donor, the energy barriers
are 10-15 kcal/mol lower for the reactions involving arsenic substrates as arsenate donors.
The solvent calculations (Table 2) also show a trend similar to the gas phase calculations. The
products become relatively less stable with subsequent replacement of phosphorus by arsenic. The
reactions with phosphorus substrates are more favorable by ~ 1.0 - 5.0 kcal/mol, whereas the
activation barriers are much lower for the arsenic substituted reactions by nearly ~ 15.0 kcal/mol.
Both, gas phase and solvent phase calculations reveal that arsenic can be a potential substitute of
phosphorus in biomolecules.
In neutral solution, as ATP is ionized and exists mostly as ATP4−
, we also studied the reaction
with the oxyanions of H3PO4, H4P2O7, H5P3O10, CH3H4P3O10 and their arsenic analogues. The
energetics for phosphorylation of ribose by the oxyanions is shown in Table 3. In presence of
charged oxygen, the relative stability of the products increases with increase in the length of the
phosphate donor. The H-bond distances in H2PO4-, H2P2O7
2- and H2P3O10
3- are 2.55 Å, 1.82 Å and
1.79 Å, respectively. Comparing the reaction with H2P2O72-
and H2P3O43-
, H-bond of the reactant is
0.03 Å shorter for H2P2O72-
, whereas the H-bond length of the product is 0.62 Å shorter for the
reaction with H2P3O103-
. Hence, the products get more and more stabilized with the increasing chain
length of the reacting phosphate. As the H-bond distances are similar in H2P3O43-
and its methyl
derivative, the interaction energies for these reactions are also nearly the same. In the case of
solvated systems, for all substrates, PCM calculations predict a much lower exothermicity for
phosphorylation compared with the gas-phase reactions. The charged reactants have a strong
tendency to get stabilized due to solvation and therefore are less inclined to lead to the
phosphorylated product as compared to in gas phase. In addition, in phosphorylated and arsenylated
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ribose, the H-bond lengths are longer in solvent compared to in gas phase. The hydrogen bond
distances in ribose-phosphate and ribose-arsenate in gas phase are 2.43 Å and 2.58 Å, respectively.
Whereas, the hydrogen bond distances are 2.58 Å and 2.73 Å in water for phosphorylated and
arsenylated ribose, respectively. Hence, the stabilization of the products by hydrogen bonds is more
in gas phase as compared to in water.
In order to attain a better picture of the microenvironment where-in in-vivo phosphorylation
takes place, we looked at kinases, the group of enzymes involved in transfer of phosphoryl group
from a phosphate donor (usually ATP) to a phosphate acceptor. Ribokinse (RK)-like kinases are the
enzymes involved in the phosphorylation of ribose and glucose.34
Despite the diversity observed in
RK-like kinases, the substrate binding pocket has similar overall geometry.35
The phosphoryl
transfer involves the deprotonation of sugar-hydroxyl group by a catalytic base, mostly Asp residue.
The oxygen atom then attacks the γ phosphate group of ATP. The kinase members also utilize a
magnesium ion and an additional electrostatic interaction between a lysine (or equivalent arginine)
and β-phosphate of ATP. Thus, considering the conserved residues we have modeled a system
including sugar, ATP, Mg2+
, aspartic acid and lysine for QM/MM computations from a glucokinase
crystal structure (PDB id: 3ID8). Phosphorylation of both glucose and ribose were studied. The
quantum mechanics region includes the β-, γ-phosphate group, hydroxymethyl group of sugar, three
water molecules, anionic carboxymethyl group of aspartate residue and the methyleneammonium
group of lysine. We understand the importance of taking into account the conformational variability
of enzymes. However, in this work we restrict to optimizing the reactants and products by
consecutive replacement of phosphorus with arsenic. Our present aim is not to calculate accurate
absolute energies, but to understand the effect of incorporation of arsenic on phosphorylation,
relatively. In future, a more in-depth study of structures taken from MM and QM/MM molecular
dynamics simulations would be performed to get a better understanding of the kinetics of the
reaction. Table 4 shows the relative stability of the sugar phosphorylated by ATP and its arsenic
analogues. The ΔE and ΔG values calculated by QM/MM computations also follow the same trend
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as revealed by the QM calculations. The overall reaction of phosphate transfer from ATP to glucose
is exothermic by -16.4 kcal/mol. About 2.0 kcal/mol lowering in ΔE is observed on substitution of
phosphorus by arsenic suggesting that arsenic can be a perfect substitute for phosphorus.
As the catalytic activity of a given molecular environment depends on the activation energy
barrier, the TS structures were also analyzed. The TS (Figure 2) indicates a partially associative
SN2-like mechanism involving TS with a length of axial phosphorus-oxygen bonds - 2.43 Å and 3.2
Å and axial arsenic-oxygen bonds – 2.33 Å and 2.75 Å.36
The TS for the arsenic analogue is more
stabilized due to the interaction between the different residues as compared to its phosphorus
counterpart. The Mg-O (phosphoryl/arsenyl group) interaction distances are 2.06 Å and 1.99 Å, in
the TS for ATP and As-ATP, respectively. The H-bonding distances are also longer in the ATP
involved transition state (1.65 Å, 1.81 Å, 1.74 Å, and 2.44 Å) as compared to its arsenic analogue
(1.63 Å, 1.65 Å, 1.66 Å, and 2.01 Å). The comparably larger stabilization of the TS lowers the
energy barrier by ~ 1kcal/mol. Our model leads to a +11.4 kcal/mol barrier for ATP reaction and a
+10.2 kcal/mol activation barrier for the reaction with As-ATP. The activation barrier for
phosphorylation of ribose is calculated to be + 27.1 kcal/mol, about 16.0 kcal/mol higher than the
barrier for glucose.
Measurement of kinetic isotopic effects (KIEs) has found widespread use in mechanistic
studies of various reaction types. We specifically examine the theoretical secondary kinetic isotopic
effects (SKIEs) for protons of the three water molecules residing in the coordination sphere of
magnesium. The Arrhenius equation gives the dependence of the rate constant k on the activation
energy and temperature -
k = A exp (-Ea/kBT)
where k is the rate constant, A the pre-exponential factor, Ea is the activation energy, kB is
the Boltzman constant and T = 300K. Previously it has been shown that kinetic isotopic effect can
be evaluated by using ground- and transition-state vibrational normal modes.37-39
The infrared
spectra of the ground- and transition-states were obtained, and the fundamental frequencies were
9
assigned. Energy, Ea, is evaluated in terms of the vibrational frequencies of the three water
molecules in the transition state and the ground state (total of nine νO-H modes). Thus, the light-to-
heavy KIE is written in terms of the ratio
As 1kcal/mol = 350cm-1
where the subscript H represents water; D represents deuterated water; ν‡
is the sum of the
bending, symmetric stretching and asymmetric stretching modes of the three water molecules in the
transition state and similarly ν is the sum in the ground state. As kBT = 0.6 kcal/mol, the isotopic
effect can be determined by the following equation.
where kH/kD represents the secondary kinetic isotopic effect. The calculated SKIE’s for ATP
and ATP (As) are 5.81 and 4.73 respectively. A decrease in SKIE is observed on the substitution of
phosphorus with arsenic. The large SKIE values show that phosphate transfer involves a collective
motion of the three water molecules in order to phosphorylate the sugar moiety. The isotopic effects
therefore suggest that these three H2O molecules are important in the mechanism of phosphory-
/arseny-lation and a difference in SKIE in between P/As processes might be experimentally
determined.
CONCLUSION
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In summary, we studied the effect of substituting phosphorus with arsenic on the mechanism of
sugar phosphorylation. Phosphorylated sugar is involved in the synthesis of vital biomolecules.
Although, quantum mechanical calculations predict a marginally lower stability for arsenylated
ribose, the activation barriers for arsenylation are ~ 15.0 kcal/mol lower than the barrier for
phosphorylation. QM/MM optimizations also reveal that substitution of phosphorus by arsenic is
energetically feasible. Erb et al followed up the controversial discovery of Wolf Simon and
concluded that GFAJ-1 strain is an arsenic-resistant, phosphate-dependent organism. However, they
also report abiotic formation of some arsenylated compounds (hexose arsenates) on feeding GFAJ-1
with arsenate.40
The participation of arsenic in in-vivo processes would also include other factors,
such as, reactivity in water and the existence of enzymes which would act on arsenate substrates.
However, based on the calculations performed on our model system, we conclude that substitution
of phosphorus by arsenic in biomolecules is possible from a structural, electronic and kinetic point
of view. As the water molecules in the co-ordination sphere of magnesium are involved in
phosphate transfer, the secondary kinetic isotopic effects for the water molecules are found to be
significant. The SKIE of arsenylation is ~1.0 lesser than phosphorylation. We predict that accurate
measurement of SKIE in the arsenic based bacterial strain, GFAJ-1 can be an interesting tool to
confirm the existence of arsenic incorporation in cell cycle.
ASSOCIATED CONTENT
Supporting Information. Cartesian Coordinates, Energies, Harmonic frequencies, complete
Gaussian 09 references.
AUTHOR INFORMATION
Corresponding Author:
ACKNOWLEDGMENT
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AD and JAK thank UGC and CSIR India for financial assistance. AD thanks DST, CSIR and INSA
for partial funding.
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Tables:
Table 1 Energetics (gas phase) of β-D-deoxyribose phosphorylation by phosphoric acid,
pyrophosphoric acid, DHP and their arsenic analogues along with the free energy barrier for the
reactions at M06-2X/6-311+G(d,p) level.
Molecule ∆E
(kcal/mol)
∆E‡
(kcal/mol)
∆G
(kcal/mol)
∆G‡
(kcal/mol)
H3PO4 -13.0 - -9.8 -
H3AsO4 -11.6 - -7.5 -
H4P2O7 -7.3 34.0 -7.4 33.21
H4PAsO7 -5.4 27.7 -5.1 25.2
H4As2O7 -5.1 18.6 -6.0 18.8
H5P3O10 -14.3 25.5 -12.5 26.1
H5P2AsO10 -10.5 26.5 -8.8 25.2
H5PAs2O10 -11.0 27.3 -9.9 26.1
H5As3O10 -8.8 27.0 -7.3 25.7
CH3H4P3O10 -16.4 34.0 -15.1 35.7
CH3H4P2AsO10 -12.4 20.6 -11.0 20.4
CH3H4PAs2O10 -12.4 20.9 -11.3 20.8
CH3H4As3O10 -10.4 20.7 -9.7 20.5
15
Table 2 Energetics (solvent = H2O; PCM model) of β-D-deoxyribose phosphorylation by
phosphoric acid, pyrophosphoric acid, DHP and their arsenic analogues along with the free energy
barrier for the reaction at M06-2X/6-311+G(d,p) level.
Molecule ∆E (kcal/mol) ∆E‡ (kcal/mol) ∆G (kcal/mol) ∆G
‡ (kcal/mol)
H3PO4 -6.9 - -5.1 -
H3AsO4 -6.1 - -3.3 -
H4P2O7 -7.7 34.6 -8.4 33.2
H4PAsO7 -6.2 25.2 -6.2 23.4
H4As2O7 -6.0 20.6 -6.5 19.5
H5P3O10 -8.7 25.5 -8.5 24.2
H5P2AsO10 -6.0 24.6 -5.7 22.4
H5PAs2O10 -6.1 25.3 -6.2 24.2
H5As3O10 -4.2 24.7 -4.5 21.6
CH3H4P3O10 -10.3 33.6 -10.0 35.7
CH3H4P2AsO10 -7.4 18.3 -7.3 18.7
CH3H4PAs2O10 -7.3 23.1 -6.6 22.1
CH3H4As3O10 -5.7 17.7 -5.9 18.2
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Table 3. Energetics (gas phase and solvent = H2O) of β-D-deoxyribose phosphorylation by
oxyanions of phosphoric acid, pyrophosphoric acid, DHP and their arsenic analogues at M06-2X/6-
311+G(d,p) level.
Molecule ∆E
(kcal/mol)
∆G
(kcal/mol)
∆Ea
(kcal/mol)
∆Ga
(kcal/mol)
H2PO4-
-23.2 -20.1 -9.5 -6.8
H2AsO4 -
-21.2 -18.4 -8.9 -6.4
H2P2O7 2-
-70.0 -71.0 +1.1 -0.2
H2PAsO7 2-
-66.8 -67.7 +2.2 +1.0
H2As2O7 2-
-65.6 -66.2 +2.8 +1.7
H2P3O10 3-
-138.0 -135.5 -10.3 -8.0
H2P2AsO10 3-
-132.9 -130.2 -9.0 -7.1
H2PAs2O10 3-
-130.8 -128.3 -8.3 -6.9
H2As3O10 3-
-129.3 -126.6 -8.5 -6.3
CH4P3O10 3-
-131.2 -130.4 -7.8 -7.4
CH4P2AsO10 3-
-125.9 -124.8 -6.5 -6.5
CH4PAs2O10 3-
-124.6 -123.8 -5.5 -4.7
CH4As3O10 3-
-126.9 -124.8 -7.5 -6.3
a: PCM calculations
Table 4. Energetics of phosphorylation of glucose and β-D-deoxyribose by ATP and its arsenic
analogues (QM/MM : B3LYP/6-31G(d)/AMBER).
Molecule ∆Ea
∆Ga
∆Eb ∆G
b
ATP -16.4 -4.4 -2.1 +2.3
ATP I
-11.3 +4.0 -0.1 +6.1
ATPII
-11.1 +1.7 +1.0 +6.8
ATPIII
-15.8 -7.6 -0.8 +6.7
ATP: Adenine TriPhosphate; ATPI – ATP with one phosphorus replaced by arsenic; ATPII – ATP with two
phosphorus replaced by arsenic; ATPIII – ATP with three phosphorus replaced by arsenic. a: Glucose; b: Ribose
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Figures:
Scheme 1. Addition of phosphoric acid, pyrophosphoric acid and DHP to β-D-deoxyribose ( X=P,
As).
Figure 1. Transition state structure for transfer of phosphate from methyl-DHP to β-D-deoxyribose
at M06-2X/6-311+G(d,p).
.
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Figure 2. A) Reaction mechanism of glucose phosphorylation by ATP. B) QM/MM (B3LYP/6-
31G(d)/AMBER) optimized structures of reactant, transition state and product.