(Received for publication, August 22, 1994; and in revised form, November 28, 1994)
From the
Single and multiple site mutants of recombinant mouse
acetylcholinesterase (rMoAChE) were inhibited with racemic
7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the
resulting mixture of two enantiomers,
CHP
(O)(OC
H
)-AChE(EMP
-AChE),
were subjected to reactivation with
2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and
1-(2`-hydroxyiminomethyl-1`-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2-oxapropane
dichloride (HI-6). Kinetic analysis of the reactivation profiles
revealed biphasic behavior with an approximate 1:1 ratio of two
presumed reactivatable enantiomeric components. Equilibrium
dissociation and kinetic rate constants for reactivation of
site-specific mutant enzymes were compared with those obtained for
wild-type rMoAChE, tissue-derived Torpedo AChE and human
plasma butyrylcholinesterase. Substitution of key amino acid residues
at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and
Asp-74) had a greater influence on the reactivation kinetics of the
bisquaternary reactivator HI-6 compared with the monoquaternary
reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by
HI-6 but not by P2S. Of residues forming the choline-binding subsite,
the E202Q mutation had a dominant influence where reactivation by both
oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in
this subsite showed little involvement. These kinetic findings,
together with energy minimization of the oxime complex with the
phosphonylated enzyme, provide a model for differences in the
reactivation potencies of P2S and HI-6. The two kinetic components of
oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and
may be attributable to the relative stability of the phosphonyl oxygen
of the two enantiomers in the oxyanion hole.
Inhibition of acetylcholinesterase (AChE; EC 3.1.1.7) ()and butyrylcholinesterase (BChE; EC 3.1.1.8) by
organophosphorus (OP) esters is attributed to the formation of a
covalent conjugate between the OP moiety and the active-site serine of
the enzyme(1) . OP-inhibited cholinesterases (ChEs) (
)can be reactivated by certain oxime nucleophiles, if the
enzyme does not undergo a prior ``aging''
reaction(1, 2) . Since the discovery of powerful
reactivators of OP-inhibited ChEs,
2-(hydroxyiminomethyl)-1-methylpyridinium iodide (2-PAM; Fig. 1)(3) , and the bispyridinium dioxime
1,1`-trimethylene
bis(4-hydroxyiminomethylpyridinium)-dibromide(4) , several
reports have described the preparation, structure, and biochemical
properties of mono- and bisquaternary oximes. The limited scope of
antidotal activity of commonly used reactivators such as the
methanesulfonate salt of 2-PAM (P2S; Fig. 1) or
1,1`-trimethylene bis(4-hydroxyiminomethylpyridinium)-dibromide against
certain types of OP anti-ChE, prompted the evaluation of a new series
of oxime reactivators(5) . One such compound,
1-(2`-hydroxyiminomethyl-1`-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2-oxapropane
dichloride (HI-6; Fig. 1) is among the most potent reactivating
agents that serve as antidotes against organophosphate
toxicity(6, 7) .
Figure 1: Structures of 2-PAM, P2S, and HI-6. The syn configurations shown are in accordance with the crystal structure of 2-PAM (19) and HI-6(20) .
The effectiveness of oxime reactivators as antidotes is primarily attributed to the nucleophilic displacement rate of the OP moiety from the inhibited enzyme (Fig. 2) and varies with the structure of the bound OP, the source of the enzyme, and the oxime. In spite of three decades of progress in improving the reactivation properties of the lead compounds, structure-function relationships are not clearly understood.
Figure 2:
Chemical pathways and kinetic schemes of
the reaction of oximes with MEPQ (a), and the inhibition and
oxime-induced reactivation of ChEs (b). The structure in parentheses depicts the assumed pentacoordinate transition
state having a trigonal bipyramidal geometry. Both the nucleophile and
the leaving group occupy apical positions when assuming an in-line S2 displacement
reaction.
The elucidation of the three-dimensional structure of Torpedo AChE (TcAChE)(8) , together with kinetic and mechanistic
studies of site-directed mutants(9) , have added a new
dimension to the study of organophosphate inhibition and reactivation.
In this report we describe studies on P2S- and HI-6-induced
reactivation of wild-type recombinant mouse AChE (rMoAChE) and its
mutants inhibited with
7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ; Fig. 2) (10) . Delineation of amino acid residues that
are important for reactivation highlight several aspects of the
mechanism by which oximes enhance displacement of an OP from EMP-ChEs
and provide an explanation for differences between the reactivation
potency of HI-6 and P2S. In addition, owing to chirality of the
phosphorus in MEPQ and the potent anti-ChE activity of both of its
enantiomers(10, 11) , the inhibited enzyme consists of
two enantiomeric components, EMP-ChE, and
EMP
-ChE, that are amenable to analysis of the
stereospecificity of the reactivation process.
Wild-type and mutant
rMoAChEs were prepared as described previously (12, 13) . The cDNA insert encompassing exons 2, 3, 4,
and 6 was placed behind the human cytomegalovirus promoter. Most of the
expression plasmids exist as stable transfectants in human embryonic
kidney (HEK-293) and Chinese hamster ovary (CHO-K1) cells. They secrete
into the medium hydrophilic enzyme which was concentrated by
ultrafiltration for the kinetic studies. Torpedo californica AChE, wild-type mouse AChE, and some of the mutant enzymes were
purified by affinity chromatography as described
previously(14) . HuBChE was purified by
procainamide-Sepharose 4B gel affinity chromatography.
One mg of pure enzyme contained approximately 11 and 14 nmol of active
sites of BChE and AChE, respectively. Inhibition and reactivation
experiments were carried out in enzyme solutions prepared in
microfiltered 0.05% bovine serum albumin containing 25 mM phosphate buffer, pH 7.8, at 25 °C.
where E = 100 E
/E
.
Since reactivation profiles of EMP-ChEs displayed marked deviations from a first-order approach to reactivation of a single reactivatable species, was used to determine the best-fit values of the following parameters:
where E and E
are the
percent-amplitudes of two reactivatable forms of MEPQ-inhibited ChE and
the parameters k(1) and k(2) are the corresponding
fast and slow pseudo first-order rate constants of the reactivation of E
and E
, respectively. Ratios
of E
/E
ranged between 0.8 and
1.2. In those cases where nonlinear regression did not converge due to
insufficient data points, curve fittings were processed assuming an E
to E
ratio of 1. To fit the
data to a single exponential curve, E
in was set to zero. A statistical F test was used to
compare single and biexponential nonlinear regression fits to the data. E
, E
, k(1), and k(2) were determined by computer iterations to give best-fit
values for these parameters. Nonlinear regression and statistical F test analyses were performed by Graphpad Inplot Software, version
4.01, 1992 (GraphPad Software, Inc., San Diego, CA).
Modelling
was done in vacuo, with the dissociation state of ionizable
groups set equivalent to pH 7.8. First, the O-ethyl
methylphosphonyl group was covalently attached to the O of the active-site serine and the conformation of the conjugate
minimized. By the initial placement of the oxygen of P=O bond in
the oxyanion hole, the phosphonyl moiety is susceptible to an
``in-line'' S
2 displacement. Oxime
groups were ionized and then partial charges of oximes calculated using
the MOPAC module of InsightII. Then 2-PAM and HI-6 were minimized in
the model of the phosphonylated enzymes leaving specified residues to
rotate freely.
The assumed complex of the transition state for the
reactivation of EMP-AChE by HI-6 (Fig. 2b) was analyzed by initially constraining the
three putative hydrogen bonds between the phosphonyl oxygen and the
oxyanion hole to distances <2.7 Å. Water molecules found in
the gorge in the crystal structure were included. Amino acid side chain
residues surrounding the EMP conjugate and EMP itself were allowed to
rotate, whereas the peptide backbone and distal side chains were fixed.
The pentacoordinate complex containing the covalently attached HI-6 to
the phosphorus was initially minimized by 100 iterations, equilibrated
by running dynamics at 300 K for 100 fs and then at 700 K by 50
subsequent runs of 50 fs. The seed number of the random number
generator is changed after each 50-fs run. Data on possible structures
were collected from the last 0.5 ps of each run. These structures were
then slowly cooled using steps of 50° from 700 to 300 K. Then, the
system was minimized with 1000 iterations using a conjugate gradient
method. A dielectric constant of 4.0 was used. The above algorithm was
created to avoid falling into local energy minima. This algorithm
raises the temperature of the EMP-AChE conjugates to an equilibrium
state where upon cooling they should approach global energy minima.
RESULTS ()
To compare reactivity of the actual
nucleophiles, bimolecular rate constants were corrected by dividing k by the fraction of the oximate anion at pH
7.8, [1 +
10
]
.
Using pK
values 8.07 ± 0.02 (2-PAM) and
7.47 ± 0.01 (HI-6), k
increased to 98.9
and 49.9 M
min
,
respectively. Fluoride anion is approximately 1.8 and 3.6-fold less
potent as a nucleophile than the oximate forms of HI-6 and 2-PAM,
respectively.
Scheme I:
Scheme I. Equilibria for reversible
inhibition of AChE by oximes. The [AChEoxime
ATC]
complex is assumed not to lead to
hydrolysis.
Plots of 1/Vversus 1/S were linear, and, for most
of the enzyme preparations, the intersections from Lineweaver-Burk
plots occurred in the upper left quadrant (not shown). These
observations suggest that both 2-PAM and HI-6 are likely to exhibit
linear mixed-type inhibition in accordance with Fig. SI(17) . For the series of enzymes, dissociation
constants of HuBChEoxime and rMoAChE-W286R
HI-6 could not be
defined due to significant deviations from simple equilibrium schemes
for reversible association of inhibitor with an enzyme. The high
concentrations of ATC required to saturate mutant W86A enzyme (
)precluded an accurate determination of K
and
K
for either oxime.
The
dissociation constants of 2-PAM (Table 1) for the mutants tested
were only moderately perturbed relative to wild-type rMoAChE. The
largest destabilization was observed with Y337A and W86F as reflected
by an approximately 6-7-fold increase in the corresponding K compared with that of wild-type enzyme.
Replacement of Tyr-337 by phenylalanine produced a mutant with the same
aromatic amino acid residues within the active-site gorge as TcAChE. K
values for Y337F and TcAChE were virtually
equivalent. These findings are consistent with previous observations
that established a role for aromatic residues at positions 337 (13, 21, 22) and 86 (21, 22, 23) in the stabilizing interactions
of quaternary ammonium-containing ligands within the active-site gorge.
However, the influence of aromatic side chains at these positions is
relatively small. Similarly K
of F295L
2-PAM
decreased less than 3-fold and single (W286R) and triple
(W286A/Y72N/Y124Q) mutations at the entrance to the gorge increased the
dissociation constants of 2-PAM 2.7- and 4.7-fold, respectively.
As
summarized in Table 2, K of HI-6 for
wild-type rMoAChE is 4-fold lower than that of 2-PAM, suggesting that
the second pyridinium ring of HI-6 contributes less than 1 kcal/mol to
the stabilization of the bisquaternary oxime-enzyme complex relative to
the monoquaternary pyridinium oxime. Replacement of Tyr-337 by alanine
increased HI-6 dissociation constant 8-fold compared to the wild-type
enzyme, whereas K
of HI-6 with E202Q, F295L, and
wild type rMoAChE were essentially equivalent. The three aromatic amino
acid residues at the entrance to the gorge have been shown to
constitute part of the peripheral anionic site for bisquaternary
ligands(13, 24) . Their replacement with the residues
found in BChE increased K
of the association of
HI-6 with mutant W286A/Y72N/Y124Q only 6.5-fold relative to wild-type
rMoAChE.
In Fig. SI, K
is the
dissociation constant for the AChE
oxime
ATC complex. If
association of substrates decreases the affinity of the oxime relative
to the free enzyme, then
should be >1. Whenever plots of V
versus [oxime] gave straight lines,
K
was calculated from the intercept on the x axis. Both
2-PAM and HI-6 displayed
values of 1.5 to 6 with the following
rank order: for 2-PAM, TcAChE
wild-type rMoAChE > W286R
W86F > E202Q
triple mutant; for HI-6, wild-type rMoAChE >
triple mutant
E202Q. Thus, binding of substrate to AChEs
moderately increased the dissociation constant of either oxime. The
relative destabilization of the ternary complex of substrate, oxime,
and enzyme complex is in agreement with the dissociation constants
determined for the corresponding EMP-AChE
oxime complexes (see
below).
Rates of spontaneous reactivation of
EMP-ChE conjugates were slow; all
were less than 0.5% h
, with the exception of
EMP
-W86A which was slightly
increased to 0.8% h
. The extent of reactivation did
not differ significantly for preparations that were allowed to incubate
for 1-24 h at 25 °C before the addition of reactivator. The
almost negligible spontaneous reactivation and aging reactions simplify
evaluation of the kinetic profiles. The absence of competing processes,
together with the high bimolecular rate constants for the inhibition of
AChE (11, 25) and HuBChE (11) by MEPQ,
resulted in rapid formation of an inhibited enzyme with defined species
that could be transferred immediately to reactivation buffer. It should
be pointed out that the concentration of the product 7-HQ in the
reactivation medium is equal to the active site concentration of the
inhibited enzyme (Fig. 2b). To ascertain whether 7-HQ
interferred with reactivation, up to 5 times the initial concentration
of MEPQ-inhibited enzyme in reactivation medium of 7-HQ was added to
reactivation buffer. Increased 7-HQ did not significantly affect
reactivation rate constants.
Figure 3:
Time course of reactivation of MEPQ- and
paraoxon-inhibited ChEs, by 1 mM 2-PAM (P2S). Broken and solid lines were fitted to the open squares using mono- and biexponential equations, respectively. A,
MEPQ-inhibited wild-type rMoAChE, , 2 nM;
, 5
nM;
, 20 nM. B, MEPQ-inhibited
HuBChE,
, 3.4 nM;
, 10 nM. C, paraoxon-inhibited wild-type rMoAChE,
, 2 nM;
, 10 nM. D, paraoxon-inhibited HuBChE,
, 0.5 nM;
, 34 nM. The ordinates show percent of maximal reactivation after 48
h.
At concentrations of
EMP-ChE
, ranging
between 2 and 20 nM of
EMP
-AChE and between 3.4 and 10
nM EMP
-BChE the kinetics
of oxime reactivation were virtually identical. Hence, inhibition by
reactivation product in these concentration ranges is not likely to
influence reactivation rates.
Figure 4:
Semilogarithmic plots of %
EMP-rMoAChE versus time of incubation with
various nucleophiles. Solid lines were fitted to the data
assuming two distinguishable reactivatable components. The broken
lines were fitted to data (n = 5-6 each)
that represent the slow component, in accordance with a single species
of reactivatable enzyme, and extrapolated to t = 0 to
calculate the ratios of the two reactivatable components. A,
0.9 mM 2-PAM (P2S), [fast]/[slow] =
0.89; B, 0.12 mM HI-6,
[fast]/[slow] = 0.96; C, 10 mM NaF, [fast]/[slow] = 1.2. The ratios of k(fast) to k(slow) were 5.5, 7.1, and 8.0 for 2-PAM,
HI-6, and NaF, respectively.
These findings are satisfactorily explained by the presence of two kinetically distinguishable EMP-ChE enantiomers. However, since it was observed for bovine erythrocyte AChE that the achiral inhibitor, paraoxon, also produces more than one class of reactivatable species (26) , it was necessary to compare the reactivation profile of paraoxon- and MEPQ-ChE conjugates under the same experimental conditions. Results are depicted in Fig. 3(panels C and D). Indeed, for both achiral paraoxon-inhibited rMoAChE and HuBChE, the biexponential equation improved the fit only slightly, but significantly, better than a single component model.
Although the deviations from kinetics of a homogenous class of
inhibited enzyme species were substantially larger for the
EMP-ChE conjugates compared to
similar preparations using paraoxon, we could not ascertain the basis
of an intrinsic, but small, contribution to the deviation from
monoexponential decay function for the achiral O,O`-diethylphosphoryl-AChE conjugate. Therefore, we
categorize the two phases in as fast and slow components
of oxime-induced reactivation, and k(1) and k(2) are
the corresponding first-order rate constants of the fast and slow
components, respectively, in the presence of large stoichiometric
excesses of the reactivator.
The mathematical solution for the kinetic scheme of the reactivation depicted in Fig. 2b is:
where k is either k(1) or k(2), and K`
= (k
+ k`
)/k
. Assuming k
k`
, K`
is approximated by k
/k
which is the
corresponding dissociation constant of the complex EMP-ChE
oxime.
The individual constants k`
and K`
were determined by nonlinear regression
analysis of the data shown in Fig. 5, according to .
The bimolecular rate constant of reactivation (k
)
was obtained by dividing k`
by K`
.
Figure 5:
Representative plots of kversus [oxime] for reactivation of
EMP
-AChE. Lines were fitted to the data
in accordance with except for panel E that was
fitted to . The left- and right-hand side
panels of each pair show the fast and the slow rate constants,
respectively. A and B, P2S with wild-type rMoAChE; C and D, 2-PAM (P2S) with W86A; E and F, HI-6 with W286R; G and H, HI-6 with
W286A/Y72N/124Q.
In several cases (Fig. 5, panel
E) the plots of kversus [oxime] were linear rather than asymptotically
approaching a constant value. In this situation, the individual
component constants could not be resolved. Assuming K`
[oxime], is approximated by the
following expression:
Thus, k = k`
/K`
was obtained from
the slopes of straight lines constructed by plots of k
versus [oxime].
Table 1and Table 2summarize the kinetic constants of the reactivation of
EMP-ChEs by 2-PAM and HI-6, respectively. The last column also gives
the bimolecular rate constant for the reactivation by the oximate ions,
RCH=N-O(k
). In
general, dissociation constants of the reactivators for
EMP
-AChEs (K`
) are increased compared with
nonphosphonylated enzyme (i.e.K
<K`
). The
magnitude of destabilization of the inhibited enzyme-oxime complex was
in the range obtained for the ratio
K
/K
, an observation
that is consistent with the interpretation of
. Thus, the presence
of bound ATC or its reaction product and conjugated EMP decreased the
affinity of the oximes to an equivalent extent.
Replacement of the
-electron-rich indole side chain of Trp-86 by alanine decreased
only slightly (1.2-2.3-fold) and moderately (2.2-5.1-fold) k
(slow) of 2-PAM and HI-6, respectively,
compared with wild-type phosphonylated rMoAChE. Furthermore, k
(fast) of both 2-PAM and HI-6 were
slightly enhanced with the EMP-Y337A conjugate relative to the
corresponding reactions with wild-type rMoAChE. These findings suggest
that Trp-86, and Tyr-337 play only a limited role in binding the oxime
in a conformation suitable for reactivation of phosphonylated AChEs.
The reactivatability of the two components of
EMP-TcAChE by 2-PAM was comparable
to that of mutant Y337F. Replacement of tyrosine by phenylalanine in
rMoAChE produces a mutant that contains aromatic side chain residues of
the choline subsite identical to
TcAChE(8, 13, 28) .
One enantiomeric form of
EMP-F295L reactivated profoundly
faster than the other, which appeared resistant to reactivation. Since
the extent of maximal reactivation was independent of the time of prior
incubation of F295L with MEPQ, slow dealkylation (i.e. aging)
cannot explain the relative resistance of the second component to
oxime-induced reactivation. Similar observations were made previously
with the O-cycloheptyl methylphosphonyl-TcAChE
conjugate(29) .
Interestingly, replacement of phenylalanine
in position 295 by leucine had opposing effects on k for 2-PAM (decreased 3.7-fold) and HI-6
(increased 2.2-fold). These findings are consistent with the relative
changes observed in the affinity of the oximes (K
and K`
) for F295L (Table 1). F295L
alters the spatial constraints surrounding the O
Ser-203-bound phosphonyl moiety and thereby changes the
stereochemical requirements of the reactivation process.
Finally, k values of
EMP
-HuBChE that contains aliphatic
amino acid residues in positions homologous to 286, 124, and 72 of
AChE, revealed that 2-PAM is superior to HI-6 in reactivating HuBChE,
and the k
ratio of rMoAChE to HuBChE is
>25 with HI-6, whereas it is <2 for 2-PAM. This further
underscores the importance of the aromatic amino acids at the entrance
to the gorge of AChEs in enhancing reactivation potency of HI-6 as
compared with 2-PAM.
Figure 6:
Stereo views of the final conformations of
energy minimized 2-PAM and HI-6 in models of
EMP-AChE, EMP
-AChE,
EMP
-HuBChE, and EMP
-HuBChE.
Energy minimizations were carried out using the atomic coordinates
obtained from the crystal structure of TcAChE(8) , and HuBChE
model based on the TcAChE structure(18) . Amino acid residues
are labeled in accordance with the numbering system of rMoAChE
(EMP
-AChE) and HuBChE
(EMP
-HuBChE). Hydrogen bonds between the
phosphonyl oxygen and the amide hydrogens in the oxyanion hole are
shown by dotted lines. The hydrogen bond formed between the
hydroxyl of Tyr-124 and the etheral oxygen of HI-6 is not shown.
Selected final geometries of energy minimized 2-PAM within the
corresponding conjugates were included to illustrate differences in
steric hindrance around the P-atom. Dotted spheres are van der
Waals surfaces of CH
-P, CH
CH
O-P,
and RC=N-O
of 2-PAM modeled and energy
minimized within the corresponding EMP-AChE conjugates. The final
conformation of 2-PAM shown is one of several closely related
overlapping structures with similar energy
content.
Although the charge on the pyridinium nitrogen (N)
is delocalized(30) , it is interesting to measure distances
between N
and some of the atoms surrounding 2-PAM.
Trp-86 C
is 5.4 and 4.8 Å from N
of the R
and S
enantiomers, respectively. Both Tyr-337 C
, and
Phe-338 C
of the enantiomeric EMP-AChE conjugates are
>6.2 Å away from the pyridinium nitrogen. These distances are
in fair agreement with experimental observations showing that k
is not significantly affected by single
replacement of an aromatic side chain by aliphatic amino acids at
positions 86 and 337.
Of the two carboxylate side chains that are
projected into the gorge, the carboxylate oxygen of Asp-74 is 4.2 and
5.0 Å from the quaternary nitrogen of 2-PAM modeled in the R and S
conjugates,
respectively, whereas Glu-202 carboxylate is about 9.5 Å from
nitrogen in both enantiomers. Despite the greater distance of Glu-202
carboxylate from the pyridinium nitrogen compared with Asp-74
carboxylate, perturbation of reactivation with 2-PAM was significantly
greater with E202Q compared to D74N. We note that the distance of
either Glu-202 carboxylate or His-447 N
from the
oximate oxygen ranged from 5.8 to 8.8 Å in all
EMP
-AChE
oxime complexes.
The oxime-containing pyridinium ring of HI-6 is oriented essentially
as described above for the 2-PAM complex. The carbamoyl, C(O)NH moiety of the distal pyridinium ring is projected toward the
peripheral binding site and forms close contacts with aromatic side
chains of Trp-286, Tyr-72, and Tyr-124. Computer-simulated molecular
dynamics of the ground state (not shown) and the pentacoordinate
transition state (Fig. 7a) of
EMP
-AChE
HI-6 clearly point to
the ability of the hydroxyl of Tyr-124 to hydrogen bond to the oxygen
of the bismethylene ether moiety that connects the two pyridinium
rings. These interactions appear to restrict movements of HI-6 within
the gorge. Apparently, anchoring of the distal pyridinium moiety
results in shortening of the distances between N
of
the proximal pyridinium ring and Trp-86 C
(4.6
Å), Tyr-337 C
(3.9 Å), and Phe-338
C
(5.1 Å), compared to
EMP
-AChE
2-PAM. The model of
EMP
-AChE
HI-6 is consistent with the finding that
mutations of residues Trp-286, Tyr-72, and Tyr-124 decreased
dramatically k
of HI-6 but not of 2-PAM.
Molecular dynamics carried out by equilibration of the
EMP
-AChE
HI-6 complexes at
high temperature followed by cooling yields a dramatic difference for
the EMP
-AChE and EMP
-AChE enantiomers. In the
case of the R enantiomer the phosphonyl oxygen remains within
the oxyanion hole (Fig. 7b) while the S enantiomer assumes multiple conformations. Binding in the oxyanion
hole should enhance reactivity by lowering the energy of the transition
state and this factor could account for the different rates of reaction
of the R and S enantiomers.
Figure 7:
Molecular dynamics of the pentacoordinate
transition state between
EMP-AChE and HI-6. A
stereoview of EMP
-AChE
HI6. Geometry around
phosphorus was pentacoordinate with the oxime and Ser-203 oxygen
assuming apical positions. Shown are the results of five simulations
with heating and equilibrating at 700 K with subsequent cooling to 300
K. Simulations were constructed for R and S enantiomers of EMP. B shows an enlarged view in the
region of the pentacovalent phosphorus for the
EMP
-AChE
HI-6 transition state. Note the
fixed position of the phosphonyl oxygen. C shows an identical
simulation for EMP
-AChE
HI-6 where a large
variation of position of the residues around the phosphorus are
noted.
The overall mechanism of displacement of the phosphonyl-bound
moiety of EMP-AChE by oxime
reactivators is assumed to parallel analogous reactions with low
molecular weight organophosphate model compounds. Thus, reactivation is
expected to proceed from a tetrahedral ground state of the phosphonyl
moiety to a trigonal bipyramidal transition state (Fig. 2a)(31) . For both oximes the carboxylate
side chain of Glu-202 appears to be important for stabilizing
intermediates along the chemical pathway by an inductive electronic
influence on the phosphorus which facilitates the nucleophilic attack.
This was suggested previously to account for the diminished rates of
phosphorylation of the E202Q mutant(25) . The acquisition of a
negative charge of the transition state is likely to be stabilized by
both the amide hydrogens in the oxyanion hole and the positive charge
of the pyridinium ring. The contribution of the latter interaction to
the overall stabilization of the transition state is evident from the
high ratios of k
for 2-PAM (>125) and for
HI-6 (>500) compared with the estimated bimolecular rate constant of
the reactivation by NaF (<10 M
min
), even though the nucleophilicity of
fluoride is only 3.6- and 1.8-fold smaller than that of 2-PAM and HI-6,
respectively.
The unimolecular rate constants of the reactivation of
wild-type phosphonylated AChE by both oximes (k`, 0.25-0.58 min
) are
2500-fold higher than the estimated rate constants for spontaneous
restoration of enzyme activity. Such an enhancement suggests a decrease
of more than 4.5 kcal/mol in energy barrier, in going from initial
state of oxime-bound complex to the activated state, compared with a
general base or H
O-catalyzed reactivation. This
consideration together with results shown here reveals a specific
molecular recognition of quaternary oximes in the active-site gorge.
Considerations of energy barriers suggest that reactivation will be
favored by an in-line displacement where the nucleophile and the enzyme
occupy apical positions in the trigonal bipyramidal transition state (31) . Molecular modelling showed that the oximate oxygen of
various EMP-AChE
oxime
complexes is positioned 4.4 to 4.9 Å from the P atom, suggesting
that a change in conformation is required in order for the hydroxamate
oxygen atom to form a covalent bond with the phosphonyl moiety. This
view is supported by the observation that the ratio of dissociation
constants of the HI-6 complexes with nonphosphonylated (K
) and EMP conjugate (K`
)
of the triple mutant is 6 to 9 as opposed to a ratio of 70 for
wild-type rMoAChE. Of all the models examined, the bond angle
RCH=NO-P-O
Ser-203 that approaches
an optimal 180° for in-line displacement was found to be 169°
for EMP
-AChE
HI-6. The most likely explanation for the
overall 8-fold increase in k
of HI-6 over
2-PAM stems from a better orientation of the oximate oxygen of the
former oxime toward an apical approach to the phosphorus atom from the
face formed by three atoms and perpendicular to P-O
Ser-203 bond. Eventually, this might lead to greater
stabilization of the transition state.
Using similar arguments, the
smaller molecular volume of 2-PAM may confer to this reactivator
sufficient flexibility to accommodate itself at various overlapping
orientations within the gorge of wild-type phosphonylated AChE as well
as within a gorge of diminished aromaticity seen with BChE. This view
is supported by energy minimization yielding different final
conformations with similar energy depending on the starting positions
of the oxime within EMP-ChEs (not
shown). This flexibility may diminish the dependence of k
on structural changes with the active-site
gorge and give rise to unproductive binding conformations.
Sequence
alignments of AChE and BChE reveal that aromatic amino acids at
positions Trp-286, Tyr-124, and Tyr-72 in mammalian AChE are replaced
by aliphatic residues in BChE(13, 28) . For
EMP-HuBChE the reduced k
for HI-6 to values less than k
of 2-PAM further substantiates the
contribution of the aromatic cluster at the gorge entrance to the
enhanced potency of HI-6. Interestingly, the ratio k
(fast)/k
(slow)
for the reactivation of EMP
-HuBChE
by HI-6 was similar to wild-type rMoAChE, whereas the ratio approached
one for the mutant enzymes W286A/Y72N/Y124Q and W286R. The diminished
differences in the susceptibility of the two enantiomeric mutant EMP
conjugates to undergo reactivation with HI-6 show that productive
binding of the fast reactivatable component by peripheral residues is
significantly greater than for the slow enantiomer of wild-type
rMoAChE. Initial experiments show that replacement of aspartic acid by
asparagine at position 74 produced approximately 24- and 3-fold
decreases in k
(fast) for HI-6 and 2-PAM,
respectively. These observations suggest that the role of the conserved
carboxylate side chain of Asp-74 in stabilizing a productive
conformation of AChE
HI-6 is manifest mainly in combination with
the aromatic residues of the peripheral site.
An interesting feature
of the energy minimized EMP-AChE
HI-6 complex is
hydrogen bonding of the hydroxyl of Tyr-124 to the etheral oxygen of
HI-6. The proposed stabilization of HI-6 is consistent with a reported
decrease in reactivation potency of a congener of HI-6 in which a three
carbon methylene chain (CHS-6) was substituted for the bisoxymethylene
bridge (HS-6)(34) .
Finally, it is of interest to point out
that k of 2-PAM was reported to be
significantly larger than k
of HI-6 for the
reactivation of homologous EMP conjugate of electric eel
AChE(33) . The anomalously low potency of HI-6 as reactivator
of phosphonylated eel AChE allows one to speculate that one or more
amino acids that control the enhanced reactivity of HI-6 toward
EMP-AChE from mammals are not conserved in eel AChE.
Energy minimization of
the putative covalent enantiomeric conjugates revealed that the bond
angles P=O- - -H-N(C=O) of either
EMP-AChE (residues 121, 122, and 204) or
EMP
-HuBChE (corresponding residues 116, 117, and 199), as
well as the relevant interatomic distances, should produce three
hydrogen bonds between the phosphonyl oxygen and the backbone nitrogen
atoms of the oxyanion hole region (Fig. 6, broken
lines). By contrast, the bond lengths increase and only a single
hydrogen bond appears to stabilize the S
enantiomers of O-ethyl methylphosphonyl conjugates of
AChE and HuBChE. Furthermore, molecular dynamic simulations (Fig. 7) indicate that stabilization of the putative
P-O
of the transition state (Fig. 2b), by hydrogen bonding to the oxyanion hole, is
likely to be greater for EMP
-ChE than for the
EMP
-ChE conjugates. In the latter conjugate the
P-O
moiety of several of the lowest energy conformers
are shifted out of the oxyanion hole. These considerations predict that
the R
enantiomer is the fast reactivatable
component. The importance of the oxyanion hole in stabilizing the
phosphonyl oxygen is underscored in the recently reported crystal
structures of phosphonate complexes with lipases that are homologous to
the ChE's(39) .
The van der Waals surfaces of the
methyl group CH-P of
EMP
-AChE, as well as of EMP
-HuBChE, that are
aligned in both cases toward the oxime moiety, reveal that the oximate
oxygen should experience greater steric hindrance for its approach
toward the P atom, compared with the homologous EMP
-ChE
conjugates (Fig. 6). In the latter conjugate, CH
-P is projected toward the acyl
pocket (Phe-295, Phe-297), the methylene of the ethyl moiety that faces
the oximate oxygen is removed from the phosphorus by an oxygen ester
linkage (P-OCH
CH
),
and thereby a larger space is opened to the oxime from the side
envisaged for the nucleophilic attack. These observations are also
consistent with EMP
-ChE being the enantiomer of
MEPQ-inhibited ChEs exhibiting rapid reactivation.