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PROTEINS: Structure, Function, and Genetics 28:543–555 (1997)
Molecular Mechanic Study of Nerve Agent O-Ethyl
S-[2-(diisopropylamino)ethyl]methylphosphonothioate
(VX) Bound to the Active Site of Torpedo californica
Acetylcholinesterase
Christine Albaret,1 Stéphane Lacoutière,1 William P. Ashman,2 Daniel Froment,1
and Pierre-Louis Fortier1*
1Centre d’Études du Bouchet, DGA, Vert le Petit, France
2U.S. Army Edgewood Research, Development and Engineering Center (ERDEC),
Aberdeen Proving Ground, Maryland 21005
ABSTRACT
Herein a molecular mechanic
study of the interaction of a lethal chemical
warfare agent, O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate (also called
VX), with Torpedo californica acetylcholinesterase (TcAChE) is discussed. This compound inhibits the enzyme by phosphonylating the active site serine. The chirality of the phosphorus
atom induces an enantiomeric inhibitory effect resulting in an enhanced anticholinesterasic activity of the SP isomer (VXS) versus its RP
counterpart (VXR). As formation of the enzymeinhibitor Michaelis complex is known to be a
crucial step in the inhibitory pathway, this
complex was addressed by stochastic boundary molecular dynamics and quantum mechanical calculations. For this purpose two models
of interaction were analyzed: in the first, the
leaving group of VX was oriented toward the
anionic subsite of TcAChE, in a similar way as
it has been suggested for the natural substrate
acetylcholine; in the second, it was oriented
toward the gorge entrance, placing the active
site serine in a suitable position for a backside
attack on the phosphorus atom. This last model
was consistent with experimental data related
to the high inhibitory effect of this compound
and the difference in activity observed for the
two enantiomers. Proteins 28:543–555, 1997.
r 1997 Wiley-Liss, Inc.
Key words: serine esterase; enantiomeric inhibition; stochastic dynamics; ab initio calculations
INTRODUCTION
Acetylcholinesterase (AChE) catalyzes the hydrolysis of acetylcholine (ACh) into acetate and choline
at cholinergic nerve terminals.1,2 This enzyme
presents a wide range of molecular forms depending
on its oligomeric assembly and the way it binds to
the membrane. The catalytic subunit is well conserved among the known sequences and a high
r 1997 WILEY-LISS, INC.
degree of identity is found for those residues implicated in the active site. Since the X-ray structure of
the catalytic subunit has been solved for the Torpedo
californica enzyme (TcAChE),3 it has been possible
to get more insights into this region of the enzyme.
The active site is found at the bottom of a deep and
narrow gorge and consists of at least four domains:
(1) an esteratic subsite containing the nucleophilic
serine and the residues responsible for the transition
state stabilization, (2) an anionic subsite that accommodates the positive choline moiety of the substrate,
(3) an acyl pocket that binds the acetyl group of the
substrate, and (4) a second peripheral anionic subsite (PAS), which lines the gorge entrance and is
implicated in the allosteric modulation of the enzyme.4,5 Reversible bisquaternary inhibitors like
decamethonium are known to derive their potency
from the ability to span the two anionic subsites.6
Irreversible inhibitors generally react with the active serine to form a covalent complex.7,8 This is the
case of a wide range of organophosphorus (OP)
compounds that have been referenced as chemical
warfare agents. They are of the type R1R2P(O)X,
where X is a leaving group, and R1 and R2 are either
alkyl or ester substituents. The two-step mechanism
by which they inhibit AChE is described in Figure 1.
Phosphonylation rate constants kp have been measured for a range of such inhibitors and have been
shown to vary weakly.9–11 This is not the case of the
dissociation constants Kd, which strongly depends on
the nature of the groups surrounding the phosphorus atom and the absolute configuration about the
phosphorus of asymmetric compounds.7,11 For this
reason, most of the studies have focused on the way
these agents interact with the binding pocket.
We have been interested in one particularly toxic
agent of this family, O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate, also called VX (Fig.
*Correspondence to: Dr. P.L. Fortier, Centre d’Études du
Bouchet, DGA, 91710 Vert le Petit, France.
Received 1 November 1996; Accepted 3 March 1997
544
C. ALBARET ET AL.
Fig. 1.
Two-step inhibition pathway of AChE by OP compounds.
built: In the first, the leaving group of VX was
positioned in the anionic subsite of TcAChE similar
to that proposed for acetylcholine (model A)16,17; in
the second, this leaving group was oriented toward
the gorge entrance in a favorable position for a
backside attack (model B). Molecular dynamics were
then performed on both types of complexes, and for
each enantiomer. The difference in inhibition between these enantiomers was investigated in terms
of stabilization of VX in the oxyanion hole (Gly 118,
Gly 119, Ala 201) and reactivity of the serine toward
the phosphorus atom. For this last purpose, ab initio
calculations were also performed to look over for the
best orientation of the active serine Og with respect
to the phosphorus atom.
MATERIALS AND METHODS
Fig. 2.
(right).
Structure of ACh (left) and VX in the SP conformation
2). This molecule displays one of the highest rate
constants of inhibition,7 and the SP enantiomer (VXS)
is known to be 150 times more active than its RP
counterpart (VXR).12 VX has a thioic leaving group,
as O-cycloheptyl S-[(trimethylamino)ethyl]methylphosphonothioate and O-isopropyl S-[(trimethylamino)ethyl]methylphosphonothioate, two related
OP compounds that have been extensively studied.11,13,14 On the basis of mutagenesis experiments,
it has been recently proposed that the thiocholine
leaving group of these inhibitors would orientate
toward the gorge entrance, allowing a backside
attack on the phosphorus atom.14 This result was
quite surprising, since it had been generally assumed that cationic species bound AChE in a way
favoring close interactions with residues of the anionic subsite.6,15 Since no such mutagenesis studies
are available for VX, we tried to ascertain if it was
possible to distinguish between the two orientations
by performing a molecular mechanic study of the
TcAChE–VX complex. Therefore, two models were
All calculations were performed on a SGI Power
Indigo 2 workstation and a SGI Power Challenge
4xR8000 supercomputer.
Model of TcAChE
Starting coordinates of TcAChE were taken from
entry 1ACE in the Brookhaven Data Bank.3,18 Hydrogens were added in INSIGHT (Biosym). All Arg, Lys,
Asp, Glu, N-terminal, and C-terminal groups were
assigned ionized, except Glu 443, which is located in
a hydrophobic environment.3,19 Histidine protonation sites were assigned as in Gilson.19 The missing
fragment 484-488 was added by using the standard
Biosym library, and optimization of its geometry was
achieved in DISCOVER (2.9, Biosym). Then a 200
steepest-descent steps followed by a 200 conjugategradient steps minimization was performed, constraining all heavy atoms of the crystallographic
structure to their original positions (constant of 100
kcal/mol).
Construction of the TcAChE–VXS Complexes
VXS was built by using the program QUANTA (4.2,
MSI). It was assumed to be positively charged, as
545
INTERACTION OF VX WITH ACETYLCHOLINESTERASE
judged by the pKa (8.8) (D. Casagne, personal communication, 1995) of the tertiary nitrogen during enzyme inhibition. Charges were derived from ab initio
calculations by using the program DMol (Biosym).
Docking of the molecules was achieved in a standard
way by using QUANTA (MSI) for visualization and
CHARMm 23.2 for optimization. For this purpose,
the phosphonyl oxygen of VX was carefully placed in
the vicinity of the oxyanion hole (Gly 118, Gly 119,
and Ala 201), while the phosphorus atom was positioned near the Og atom of Ser 200. The leaving
group was oriented toward the anionic subsite for
model A and the gorge entrance for model B. A grid
on torsion angles was then performed to minimize
the electronic and steric interactions with the binding pocket. Finally, a 200 steepest-descent steps,
followed by a 200 adopted basis Newton Raphson
steps, minimization of both complexes was achieved
by keeping the protein heavy atoms fixed. The
structures obtained after these minimizations were
kept as initial conformations for the molecular dynamics.
tions of the hydrogen bonds maintaining the catalytic triad when no hydrogen bonding term was
added to the potential. Those disruptions were accompanied by a shift of the f and c angles of Ser 200
from a disallowed region of the Ramachandran plot
to an authorized one. It has been shown that the
unusual position of the nucleophile backbone f and c
angles in the Ramachandran plot was a common
feature of the a/b hydrolase fold enzymes family, and
that this particular configuration around the active
residue contributed to the folding of the catalytic
triad.24 So we considered our modification to be
pertinent for the accuracy of the molecular dynamics
in our study. The van der Waals interactions between
heavy atom acceptors or donors and hydrogen atoms
implicated in the network were decreased such that
eij for these pairs was 0.05 kcal/mol and sij was 0.4
Å,23 where eij and sij are derived from the standard
Lennard-Jones combination rules:
eij 5 Œeiej
and sij 5
si 1 sj
2
.
(1)
Construction of the TcAChE–VXR Complexes
VXR was built using the same procedure as VXS.
For each model, the leaving group and the phosphonyl oxygen were oriented in the same direction as for
the VXS. The TcAChE–VXR complexes formed resulted in a swap of the O-ethyl and methyl moieties
of VXS in the respective TcAChE–VXS complexes.
Minimizations of both VXR complexes were then
achieved in a similar way as for the TcAChE–VXS
complexes.
Ab Initio Calculations
After the two complexes with VXS were refined,
the cartesian coordinates of the atoms of Ser 200, His
440, Glu 327, and VXS were extracted from the rest
of the system. To simplify the problem, abbreviated
forms of the residues were retained for the calculation20: Cb, Og, and Hg for the serine; Cb and
imidazole ring for the histidine; Cg, Cd, and Oe for
the glutamate. Hydrogens were added to the heavy
atoms when necessary for correct hybridation. Optimization was achieved in DMol (Biosym) for the two
models by using 50 minimization steps.
Molecular Dynamics
Stochastic boundary molecular dynamics (SBMD)
were performed by using a parallelized version of the
software CHARMm 23.221 purchased from MSI. The
all-hydrogens model was used and water was represented by the TIP3P type.22 An explicit angledependent hydrogen bonding term was added to the
potential function for the atoms of the catalytic triad
implicated in the hydrogen-bond network.23 This
modification was included following different molecular dynamics performed on the enzyme alone (not
shown). The trajectories indeed revealed some disrup-
The parameters for the complex were taken from the
CHARMm parameter file except for the inhibitor for
which charges were those derived from the DMol run
(see above). For nonbond interactions, a cutoff of 11 Å
was applied with a switching function between 8 Å
and 10 Å. Dielectric constant was set to 1. The SBMD
simulation method was employed as described in
studies of lysozyme,25 ribonuclease A,26 and trypsin.27 Reaction region was defined by a 23 Å sphere
centered on Ne2 of His440. Buffer region was made
of a 2 Å layer surrounding the reaction region. The
reaction zone was filled with water molecules. All
nonhydrogens atoms i of the protein in the buffer
region were harmonically constrained, using a boundary force defined by
Fi 5
23kBTS(ri ) Dri
7Dr2i 8
(2)
where kB is the Boltzmann constant, T is the temperature, Dri is the displacement vector for atom i from
its position in the energy minimized structure (see
above), and S(ri) is a switching function that decreases from 0.5 to 0 when going from the buffer/
reservoir boundary to the reaction region.28 The
mean square displacement for atom i was calculated
by using the isotropic Debye-Waller factors Bi according to the relation:
Bi 5
8p2
37Dr2i 8
(3)
To simplify the problem, we used the average value
of B 5 19.5 Å2 calculated on the heavy atoms of the
546
C. ALBARET ET AL.
Fig. 3. Stereo views of the TcAChE–VXS in model A (top) and model B (bottom). The key
residues of the active site are drawn.
buffer region. Solvation of the active site was achieved
by using a preequilibrated sphere (25 Å radius)
centered on Ne2 of His 440. Water molecules overlapping the protein–VX complex (cutoff of 2.8 Å) were
deleted. Four successive rotations of this sphere
were applied in order to minimize the remaining
holes in the complex. For the solvent boundary
forces, a radial distribution function centered on Ne2
of His 440 was applied to the oxygen atoms.28,29 The
frictional coefficients related to the stochastic forces
were assigned as follows: 62 ps for the heavy atoms
in the protein, and 200 ps for water oxygens.29
SHAKE30 was used to constraint bonds with hydrogens, allowing a 2-fs time step. The SBMD simulation was carried out by equilibrating the system at
300 K during 10 ps. A 100-ps production dynamic
followed this equilibration. Coordinates were saved
every 0.4 ps.
RESULTS
Starting Structures
The starting structures of the TcAChE–VXS complexes used for molecular dynamics are shown in
Figure 3. Only the key residues of the anionic,
esteric, and peripheral anionic subsites are drawn.
As mentioned above, model A corresponds to a
nucleophilic attack on a face adjacent to the leaving
group (opposite to the methyl), and model B to a
backside attack. The thioic part of VX is slightly bent
in the case of model A. Distance between the phosphorus and nitrogen atoms of VX is indeed 4.29 Å for
model A and 5.21 Å for model B. This difference is
essentially due to a distorted conformation of the
leaving group around the two carbons linking the
sulfur and nitrogen atoms in model A: the S—C—C
angle is 98.5°, while the C—C—N is 95.3°. Such a
distorsion is not surprising, since two of the methyls
of the ACh thioic group are replaced by isopropyls in
the VX molecule (see Fig. 2). Both orientations show
a hydrophobic interaction between one isopropyl and
the ring of Phe 330. The thioic part is mainly
oriented toward Trp 84 in model A, while it is close to
Tyr 121 and Tyr 334, two known PAS residues,4 in
model B. However, one of the two isopropyls remains
in the vicinity of Trp 84 in this second model. As
pointed out in the Methods section, both models were
547
INTERACTION OF VX WITH ACETYLCHOLINESTERASE
TABLE I. Distances in Angstroms of O(/P)VX to N in
the Oxyanion Hole for the Four Starting Complexes
Complex
NG118–0(/P)
NG119–0(/P)
N A201–0(/P)
mod. A SP
mod. A RP
mod. B SP
mod. B RP
2.41
2.52
2.57
2.47
2.74
2.75
2.71
3.00
3.23
3.21
3.75
3.90
TABLE II. Ab Initio Calculated Distances in
Angstroms for the SP Conformation
Model
A
B
Fig. 4. Stereo views showing the interaction of VX with the acyl
pocket in model A (top) and in model B (bottom). The inhibitor is
drawn in the two conformations (VXS in black and VXR in gray).
built, assuming that the phosphonyl oxygen of VX
interacts tightly with the oxyanion hole through
hydrogen bonds. Orientation of the leaving group
was also kept for both epimers. For this reason,
differences between the TcAChE–VXS and TcAChE–
VXR complexes mainly arise from the positions of the
methyl and O-ethyl parts of the inhibitor in the acyl
binding pocket (Fig. 4). In model A, the stacking of
the leaving group against the ring of Trp 84 fixes the
conformation of the thioic chain in the anionic subsite for both enantiomers. Hence the O-ethyl moiety
hardly accommodates the acyl pocket in the VXS
conformation, as can be judged from the value of the
O—C—C angle (92.3°). In the VXR conformation,
however, the methyl group can bind this pocket,
since its orientation in the active site is similar to
that of the proposed binding site of the methyl group
of the natural substrate ACh.16,17 In the case of
model B, the leaving group of VX has much more
room to move than in model A. As a consequence the
O-ethyl part can relax more easily in the VXR
conformation (the O—C—C angle is 107.2° after
minimization), resulting in a greater shift of the
P/O vector (Fig. 4). This shift does not modify the
orientations of the isopropyl groups, which remain
identical for both enantiomers. Table I summarizes
Distance
Ne2H440
Nd1H440
OgS200
Ne2H440
Nd1H440
OgS200
Ab initio
OgS200
Oe1E327
PVX
OgS200
Oe1E327
PVX
2.63
2.47
4.41
2.56
2.45
2.58
the distances of the phosphonyl oxygen to the oxyanion hole for the energetically optimized structures
of the four TcAChE–VX complexes. The values are
in good agreement with those recently published
for the complex of TcAChE with the transition-state
analogue m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (2.9, 2.9, and 3.2 Å for residues Gly 118,
Gly 119, and Ala 201, respectively),17 except model B for
which the hydrogen bond with Ala 201 was lost during
minimization.
Ab initio Calculations
Geometry optimizations of the starting structures
were performed in order to see what would be the
optimal configuration of VX relative to the attacking
serine in both models. Such gas phase calculations
have to be carried out with care, since phenomena as
important as solvation of the active site is not taken
into account in the procedure.31 For our purpose, we
reduced the system to VX and the catalytic triad (Ser
200, His 440, and Glu 327), as we were only interested by the orientation of OgS200 with respect to the
phosphorus atom. Optimizations were run for VXS in
the two orientations, in order to reduce the calculation time. Similar results might be expected for VXR,
since the only major change is the swap of the
O-ethyl and methyl moieties. In the case of model A,
the distance from OgS200 to the phosphorus was 4.41
Å, while it was 2.58 Å in model B (Table II). The
greater value for model A is due to the specific
configuration of VX in this orientation: the sulfur
atom is indeed close to OgS200 (2.65 Å) in the starting
system, and optimization resulted in a shift of both
sulfur and phosphorus atoms. Apart from this shift,
no significant differences in the global orientation of
OgS200 with respect to the phosphorus atom, as
described in the last section, were observed. Concerning the hydrogen bonds implicated in the catalytic
triad, values near 2.60 Å and 2.46 Å were, respec-
548
C. ALBARET ET AL.
Fig. 5. Isotropic rms fluctuations for residues in the active site of TcAChE (AS, anionic subsite;
PAS, peripheral anionic subsite; AP, acyl pocket; ES, esteric subsite). The results are given for the
four SBMD simulations.
tively, obtained for the OgS200–NeH440 and the
Nd1H440–Oe1E327 distances in the two optimizations
(Table II). The latest value is close to the one of the
crystal structure (2.5 Å). Most surprising is the value
for the OgS200–NeH440 distance that is significantly
lower than the 3.1 Å value reported by Susmann and
colleagues.3 It is most likely that binding of the
inhibitor induces a compression of the catalytic triad
allowing the proton transfer between Ser 200 and
His 440 to occur.21,32
Molecular Dynamics
A molecular dynamic study was performed in
order to probe the behavior of the four complexes.
The quality of the SBMD simulation was assessed by
comparison of the root-mean-square (rms) fluctuations of the main residues implicated in the active
site from the dynamics with the fluctuations from
the crystal structure. The results for the four simulations are shown in Figure 5. Though the rms fluctuations are a bit larger in the dynamics, they remain
close to those of the crystal structure. The largest
differences affect the trajectory of the TcAChE–VXS
complex in model A for which residues of the este-
ratic subsite (ES) present greater fluctuations. This
is due to the fact that the O-ethyl part of VX hardly
accommodated the acyl pocket during the simulation
and thus moved in the vicinity of the active serine,
inducing larger motions of residues Gly 118, Gly 119,
Ser 200, Ala 201, and His 440. It must be noted that
in the four simulations residues belonging to this
pocket presented few fluctuations. This suggests
that the acyl pocket is minimally flexible, the Phe
288, Phe 290, Trp 233, and Phe 331 aromatic rings
restricting the size of the substituents able to accommodate it. By contrast, residues of the anionic subsite were found more mobile. In particular, Trp 84
and Phe 330 presented greater fluctuations in simulations of model B. Trp 84 belongs to the V loop of
TcAChE,3 which is implicated in the allosteric modulation of the enzyme.33 Thus, the mobility of this
residue is not surprising. Phe 330 ring rotated to
stack against the leaving group of VX. Residues of
the peripheral anionic subsite (PAS) were less perturbated in the simulations of model B than in the
simulations of model A. For instance, hydrogen bond
between the hydroxyl hydrogen of Tyr 334 and Od1 of
Asp 72 was well conserved during both dynamics of
549
INTERACTION OF VX WITH ACETYLCHOLINESTERASE
Fig. 6. Time series from the four trajectories for the distances between OgS200 and Ne2H440 (top)
and between Nd1H440 and Oe1E327 (bottom). Model A is depicted in gray, model B in black.
TABLE III. Average Values and rms Fluctuations in Angstroms for the Distances in the Catalytic Triad
7r8
Model
A
B
Distance
Ne2H440
Nd1H440
Ne2H440
Nd1H440
OgS200
Oe1E327
OgS200
Oe1E327
rms
rmax
rmin
SP
RP
SP
RP
SP
RP
SP
RP
2.94
2.80
2.55
2.62
3.03
2.93
3.37
2.62
0.11
0.12
0.07
0.16
0.15
0.09
0.07
0.14
2.69
2.56
2.39
2.46
2.66
2.72
2.99
2.38
3.29
3.13
2.87
2.81
3.41
3.17
3.84
3.02
model B, although these two residues remained close
to the leaving group of VX. Deviation of Tyr 121 in
the TcAChE–VXR complex dynamic in model A is due
to the proximity of the O-ethyl moiety of VX in this
particular conformation. In all trajectories, the catalytic triad was nearly conserved (see Fig. 6 and Table
III). This suggests that the modifications we introduced in the potential function of CHARMm in order
to parametrize the catalytic triad hydrogen bonds
were relevant. The greater fluctuations were observed for the OgS200–NeH440 distance in the trajectory of the TcAChE–VXR complex in model B. As for
the simulation of the TcAChE–VXS complex in model
A, this perturbation is due to the motion of the
O-ethyl part of VX. The specific mode of binding of
VX in model B makes that this moiety in VXR makes
close van der Waals contacts with the OgS200 atom.
This resulted in a weakening of the OgS200—NeH440
hydrogen bond during the simulation.
We looked forward to the interaction of the
phosphonyl oxygen of VX with the oxyanion hole
(Gly 118, Gly 119, Ala 201). The corresponding
hydrogen bonds network has been indeed reported to
account for more than 30% of the free energy in the
transition-state stabilization of the TcAChE–TMFPA complex.34 In all simulations the interaction
with Ala 201 was almost absent (see Fig. 7 and Table
IV), suggesting a minor role for this residue in the
550
C. ALBARET ET AL.
Fig. 7. Time series from the four trajectories for the distances between N of Gly 118 (top), Gly
119 (middle), Ala 201 (bottom), and the phosphonyl oxygen of VX. Model A is depicted in gray,
model B in black.
recognition process of VX. The TcAChE–VXS complex dynamic in model B was particularly satisfactory since the two other hydrogen bonds with NG118
and NG119 were conserved. This was not the case in
the three remaining trajectories for which at least
one of these two hydrogen bonds was lost during the
simulation. For the TcAChE–VXR complex in model
B, NG118 remained far from the phosphonyl oxygen
while NG119 interacted closely with it. In the case of
model A, hydrogen bonds with NG118 and with NG119
were, respectively, broken in the dynamics of the
TcAChE–VXS and the TcAChE–VXR complexes. Each
of these hydrogen bonds remained weak. In conclusion, VXR in model B was less stabilized in the
oxyanion hole than VXS, whereas similar behaviours
were observed for both conformations in model A.
Concerning the nucleophilic attack, no significant
modifications in the global orientation of each enantiomers were observed during the four simulations
(Fig. 8 and Table V). The value of the OgS200-P–S
551
INTERACTION OF VX WITH ACETYLCHOLINESTERASE
TABLE IV. Average Values and rms Fluctuations in Angstroms for the Distances in the
Oxyanion Hole
7r8
Model
A
O(/P)VX
B
O(/P)VX
rms
rmin
rmax
Distance
SP
RP
SP
RP
SP
RP
SP
RP
NG118
NG119
NA201
NG118
NG119
NA201
3.38
2.75
3.84
3.09
2.56
4.02
3.08
3.60
3.66
4.04
2.74
3.84
0.20
0.10
0.10
0.10
0.06
0.13
0.09
0.13
0.10
0.17
0.10
0.13
2.73
2.53
3.55
2.97
2.42
3.71
2.81
3.22
3.36
3.64
2.51
3.59
3.82
3.07
4.11
3.50
2.75
4.45
3.32
3.91
3.90
4.50
3.03
4.11
Fig. 8. Time series from the four trajectories for the distance between OgS200 and the
phosphorus atom of VX (top) and for the angle of the nucleophilic attack (bottom). Model A is
depicted in gray, model B in black.
angle in the two dynamics of model A, around 50°,
may appear curious. In fact, the leaving group of VX
is perpendicular to the attacking nucleophile, but
the distorded conformation of the chain makes that
the OgS200-P–S angle is 30° lower than the 90°
expected value for an adjacent attack. In the case of
model B, Ser 200 remained in a favorable position for
a backside attack for both enantiomers (OgS200-P–
S < 170°). However, the attacking oxygen was ,3Å
farther from the phosphorus atom of VX in the
TcAChE–VXR complex simulation, the value for this
distance in the TcAChE–VXS complex being very
close to the ab initio calculation result.
DISCUSSION
The chiral nature of the complexes of asymmetric
OP compounds with AChE has long been a subject of
interest (see the extensive review of de Jong et al.7).
The great differences observed in the inhibition
constants and toxicity for the two enantiomers of a
552
C. ALBARET ET AL.
TABLE V. Average Values and rms Fluctuations in Angstroms for the Distance Between
the Attacking Oxygen and the Phosphorus Atom of VX
7r8
Model
A
B
Distance
OgS200
OgS200
PVX
PVX
rms
rmax
rmin
SP
RP
SP
RP
SP
RP
SP
RP
4.86
3.02
3.90
4.84
0.11
0.11
0.14
0.11
4.62
2.74
3.42
4.54
5.18
3.34
4.39
5.09
same OP compound seems to be related to structural
determinants of the AChE active site at the binding
stage.11,14,35 There are indeed little changes in the
phosphonylation rate constants for the compounds
for which they have been measured.9–11 The elucidation of the X-ray structure of Torpedo californica
AChE (TcAChE)3 has allowed investigations of structure–activity relationship by molecular modeling.4,15,32,33,35–38 This technique can give useful insights in the inhibition pathway of this type of
compounds, since the Michaelis complex cannot be
observed due to the fastness of the reaction of
phosphonylation. We have been interested in the
inhibitory properties of O-ethyl S-[2(diisopropylamino)ethyl]methylphosphonothioate, also called VX.
This asymmetric OP compound belongs to a family
containing the most powerful chemical warfare
agents ever synthesized.12 The tetrahedral arrangement about the phosphorus atom makes it a good
candidate for a transition state analogue. Since the
structure of a complex of TcAChE with such a
transition state analogue (m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone, also called TMFPA),
has recently been solved,17 some assumptions concerning the structural elements of importance in the
catalysis have been confirmed. Among them the
existence of a tripartite hydrogen-bond network including Gly 118, Gly 119, and Ala 201, responsible for
the stabilization of the carbonyl oxygen in the transition state has been assessed. It has also been emphasized that the quaternary ammonium of this compound could be stabilized by electrostatic interactions
with p electrons of some aromatic residues belonging
to the anionic subsite among which Trp 84 seems to
play a crucial role. Such interactions had been
predicted before the elucidation of the crystallographic structure.39,40 It is also known that replacement of the equivalent tryptophan in the human
enzyme (HuAChE), Trp 86, by an alanine significantly alters its reactivity toward a variety of charged
substrates or inhibitors while there is little effect on
their noncharged homologues.15
Our study of the complex of TcAChE with VX
suggests that this agent binds to the enzyme in a
different way from these compounds. In placing the
bulky charged leaving group in the anionic subsite
(model A), no major difference could be found in the
simulations of the TcAChE–VXS and TcAChE–VXR
complexes allowing to explain the difference of activ-
ity of the two epimers. On the other hand, when
placing the leaving group of VX in the gorge entrance, the enantiomers exhibited a quite different
behavior with respect to the oxyanion hole. In the
case of VXS, the phosphonyl oxygen was found to
make close hydrogen bonds with Gly 118 and Gly 119
during the 100 ps simulation, Ala 201 being less
implicated in the stabilization of the complex. By
contrast, simulation of the TcAChE–VXR complex
revealed that the O-ethyl moiety of VX could not
accommodate the acyl pocket and thus pushed the
P/O apart from the oxyanion hole. This resulted in
the break of the hydrogen bond with Gly 118, inducing most probably the lesser activity of VXR in
inhibiting AChE. For this reason, we believe this
model of binding (model B) more consistent than the
first one (model A). Another evidence argues for
model B: the leaving group is indeed oriented in a
suitable direction for a backside attack. This is the
most favorable case for such a nucleophilic displacement since the reaction can proceed by a direct
in-line mechanism. Ab initio calculations showed
that binding of VXS in this orientation resulted in a
compression of the catalytic triad,20,32 while the
nucleophilic oxygen and the phosphorus were found
to come closer. This compression was also observed
in model A, but to a lesser extent (see Table II).
However, the nucleophilic oxygen and the phosphorus remained far from each other in this model,
making the reaction difficult to achieve. Moreover,
the nucleophile should enter the tetrahedron at a
face adjacent to the leaving group, a very unfavorable case, since the pentacoordinate intermediate
would have to undergo at least one Berry pseudorotation41 (or turnstile rotation42) before the release of
the leaving group.43 Though the intrinsic energy
barrier for this isomerisation is relatively low (5–10
kcal/mol), this represents a supplementary energy
cost to be provided by the enzyme during the reaction.44 We can also note that in the case of enzymes
catalyzing phosphoryl transfers, such a mechanism
has never been evidenced.45
The unusual orientation of the cationic leaving
group of VX in the active site should not surprise. It
has been indeed already evidenced for two other
compounds of the alkyl methylphosphonothioates
family (O-cycloheptyl and O-isopropyl S-[(trimethylamino)ethyl]methylphosphonothioate) using mutagenesis of mouse AChE residues belonging to the
INTERACTION OF VX WITH ACETYLCHOLINESTERASE
553
Fig. 10. Model of interaction of methylphosphonothioates with
AChE. The case of the SP configuration is depicted. The O-alkyl
and methyl moieties must be swapped to obtain the RP configuration.
Fig. 9. Closeup of the active site of the TcAChE–VXS complex
average structure in model B. Residues within a distance of 3.5 Å
from any atom of VX are drawn.
acyl pocket.14 More recently, Asp 74 (72 in TcAChE),
a key residue of the peripheral anionic subsite of
mouse AChE, has been shown to be responsible for
the high reactivity of these two compounds. This
residue interacts with the quaternary ammonium of
both inhibitors through coulombic forces.44 These
forces represent the main contribution in the electrostatic potential, since the orientation of the quaternary ammonium is not adequate for optimizing the
cation-p interactions with the indole ring of Trp 86
(84 in TcAChE).44,46 Nevertheless, as the O-alkyl
moieties of both compounds point toward the anionic
subsite in the SP configuration, this tryptophan can
interact with these substituents through London
dispersion forces. This alternate mode of binding has
been highlightened by Nair and colleagues47 using
quantitative structure–activity relationship (QSAR)
calculations on a series of trifluoroketone transitionstate analogues bound to the active site of TcAChE.
In our study, Asp 72 was found to remain relatively
close to the nitrogen atom of VX in both simulations
of model B. Average values of 6.07 Å and 6.68 Å were,
respectively, obtained for the Oe1D72–NVX distance in
the TcAChE–VXS and TcAChE–VXR dynamics, allowing a substantial electrostatic contribution of Asp 72
to the binding of the inhibitor. Role of the anionic
subsite aromatic residues in the recognition process
might not be depreciated otherwise. Indeed, Trp 84
appears to remain close to the isopropyls of VX
during both dynamics, while Phe 330 was found to
stack against the leaving group. These two close
interactions are depicted for the TcAChE–VXS complex average structure on the MOLSCRIPT plot48 of
Figure 9. Thus it is most likely that this particular
compound enhances its activity by optimizing London and cation-p interactions of its leaving group
with the aromatic residues of the anionic locus, and
by interacting electrostatically with Asp 72.
CONCLUSION
Using molecular dynamics together with ab initio
calculations, we were able to propose a model of
interaction of O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate (VX) with TcAChE consistent with the high inhibitory effect of this compound
and the difference of activity observed for its epimers.
The leaving group points toward the gorge entrance,
favoring a backside attack of the active serine on the
phosphorus atom. This orientation has already been
evidenced for two other O-alkylmethylphosphonothioates compounds.14 Therefore, it is most likely that
inhibitors related to VX adopt the orientation depicted in Figure 10 when binding to the active site of
the enzyme. Since steric limitations of the acyl
pocket may prevent the accommodating of too bulky
O-alkyl moieties in the RP conformation, this model
is probably valid for a narrow range of compounds for
which the enantiomeric effect is significant.
ACKNOWLEDGMENT
We thank Dr. F. Bontems for helpful discussions
related to molecular dynamics.
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