From the Molecular Neuroscience Program,
§ Department of Molecular Pharmacology and Experimental
Therapeutics,
Mayo Clinic Cancer Center, and ** Tumor Biology
Program, Mayo Foundation for Medical Education and Research, Rochester,
Minnesota 55905 and the ¶ Eppley Institute and Department of
Biochemistry and Molecular Biology, University of Nebraska Medical
Center, 986805 Nebraska Medical Center, Omaha,
Nebraska 68198-6805
Received for publication, July 26, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Butyrylcholinesterase (BChE) is important in
cocaine metabolism, but it hydrolyzes ( Cocaine overdose is a leading cause of death among urban-dwelling
young adults (1, 2). A new idea for treating cocaine overdose is to
accelerate metabolic clearance by administering an appropriate
hydrolase. Plasma butyrylcholinesterase
(BChE)1 can hydrolyze cocaine
to ecgonine methyl ester and reportedly accounts for all the
blood-borne cocaine hydrolysis activity in humans (3-5). Pretreatment
with human BChE provides rats with substantial protection against
cocaine-induced cardiac arrhythmia, hypertension, and locomotor
hyperactivity (6). Exogenous BChE is also able to rescue rats given an
overdose of cocaine (7). However, the kcat of
BChE for ( Theoretical 3D structures of BChE (10-12), of its complex with cocaine
(9), and of the cocaine molecule itself have all been reported
(13-16). Nevertheless, the literature provides no explanation of how
cocaine forms its Michaelis-Menten complex with BChE or why BChE
hydrolyzes the unnatural stereoisomer much faster than the natural
cocaine although their Km values are similar (9, 17,
18). Addressing these questions with a computational approach should
reduce the need to construct and evaluate many mutant enzymes in a
random search for an effective cocaine hydrolase. We chose to predict
the Michaelis-Menten complex of BChE with (+)- and ( BChE and Cocaine Structures--
The natural and unnatural
cocaine structures in the protonated state (see Fig. 1) were built by
employing the PREP, LINK, EDIT, PARM, and SANDER modules of the AMBER
5.0 program (21) with the force field by Cornell et al. (22)
and additional force field parameters provided in the supporting
information. The RESP charges of these molecules were generated by
calculating electrostatic potentials using the GAUSSIAN 98 program (23)
with the HF/6-31G*//HF/6-31G* method followed by a two-stage fitting
using the RESP module of the AMBER 5.0 program (24). All such charges
are available in the supporting information.
The BChE structure was modified from a theoretical 3D structure of
human BChE, derived by homology modeling on the basis of the x-ray
structure of Torpedo acetylcholinesterase and
confirmed by site-directed mutagenesis studies (10). The modification procedure included: 1) protonation or deprotonation of the Arg, Lys,
Asp, Glu, His, and Cys residues; 2) addition of counter ions to
neutralize the charged residues; and 3) an energy minimization of the
resulting structure. To determine the protonation state, all Arg, Lys,
Asp, Glu, His, and Cys residues were visually inspected. Asp and Glu
were treated as deprotonated unless they were located in a hydrophobic
environment. One Na+ cation was placed in the vicinity of a
deprotonated, anionic residue if this residue was more than 8 Å away
from a cationic residue. Arg and Lys were treated as protonated unless
they were surrounded by hydrophobic residues. One Cl
Specifically, in the BChE structure, Cys65,
Cys92, Cys252, Cys263,
Cys400, and Cys519 were deprotonated;
His77 and His423 were protonated;
His126, His214, His207, and
His438 were assigned as HID (N Conformational Searches--
Conformational searches were
performed for (+)- and ( Docking Studies--
All docking studies were performed by using
the EUDOC program (25, 26).2
This program systematically translates and rotates a ligand in a
putative binding pocket of a receptor to search for energetically favorable orientations and positions of the ligand. A box is defined within the binding pocket to confine the translation of a ligand. The
energy used to judge the preferred orientation and position of the
ligand is termed intermolecular interaction energy and is defined as
the potential energy of the complex relative to the potential energies
of the ligand and receptor in their free states. The potential energies
are calculated with the additive, all atom force field by Cornell
et al. (22). A distance-dependent dielectric
function was used to calculate the electrostatic interactions (28). No
cutoff for steric and electrostatic interactions was used in
calculating the intermolecular interaction energies. In this work, a
box of 5.5 × 4.0 × 10.0 Å3 was defined and
surrounded by residues Trp82, Ile442,
Glu197, Tyr128, Gly439,
His438, Met437, Ser198,
Gly115, Gly116, Gly117,
Thr120, Gln119, Asn68,
Val288, Asp70, Tyr440,
Ala328, Phe329, Tyr332,
Gly333, Trp430, Leu286,
Phe398, and Trp231 in the active site of BChE.
The translational and rotational increments were set at 1.0 Å and
10o of arc, respectively.
MD Simulations--
All MD simulations were performed by
employing the AMBER 5.0 program with the Cornell et al. (22)
force field and additional force field parameters available in the
supporting information. The MD simulation used 1) the SHAKE
procedure for all bonds of the system (NTC = 3 and NTF = 3)
(29); 2) a time step of 0.1 fs (DT = 0.001); 3) a
dielectric constant, Determination of Kinetic Constants--
Hydrolysis of the two
cocaine stereoisomers by BChE was measured over a range of substrate
concentrations between 0.3 × Km and 3 × Km. Enzymatic reaction of BChE with ( pH Dependence--
Vmax and
Km were measured as a function of pH over the range
of 5.5 to 8.5 in 0.1 M sodium phosphate buffer (pH values were determined before and after reaction to ensure that conditions were stable). Hydrolysis of (
These equations assume that only the enzyme-substrate complex
with the catalytic His438 in its neutral state can proceed
to hydrolysis. In Equation 1, Ka is the equilibrium
constant for protons binding to the enzyme-substrate complex. In
Equation 2, Ka is the equilibrium constant for
protons binding to the free enzyme. Direct estimates of these
parameters, along with associated standard errors, were calculated by
the curve-fitting routine of Sigma Plot.
Cocaine-BChE Complexes Derived from Docking
Studies--
Altogether, 15 different conformations of (
Using the EUDOC program, the active site of BChE was also docked with
15 different conformations of (+)-cocaine derived from a conformational
search. The results for the most energetically stable complex with this
unnatural cocaine isomer predicted a cation-pi interaction between the
ammonium nitrogen atom of (+)-cocaine and Trp82 (Fig. 2).
The distance between the ammonium nitrogen atom and the midpoint of the
indole ring of Trp82 was 5.1 Å. The ammonium nitrogen atom
of (+)-cocaine also favorably interacts with Glu197, in
addition to the electrostatic interactions with other anionic residues
in the catalytic gorge. The distance between the ammonium nitrogen atom
and the side chain carbonyl carbon atoms of Glu197 was 4.4 Å. In addition, the phenyl ring of (+)-cocaine weakly interacted with
Tyr332 and Phe329 (Fig. 2). Thus in many
respects the Michaelis-Menten complexes of the two cocaine isomers
appear similar. However, the distances of the midpoint of the phenyl
ring of (+)-cocaine to those of Tyr332 and
Phe329 were 6.9 and 7.7 Å, respectively. The distances of
the midpoint of the phenyl ring of ( Cocaine-BChE Complexes Refined by MD Simulations--
Separate
1.0-ns (1.0 fs time step) MD simulations of the (
In the present case, comparing all non-hydrogen, protein
backbone atoms in the initial (
The time-averaged structure of the (
The time-averaged structure of the (+)-cocaine-BChE complex derived
from the 1.0-ns MD simulation was similar to the initial structure
derived from the docking study. In contrast to the natural cocaine-BChE
complex refined by MD simulation, Phe329 and
Trp430 were not in contact with the phenyl ring of
(+)-cocaine in the time-averaged MD structure (Fig. 4). The distance
between the midpoint of the phenyl ring of (+)-cocaine and the midpoint
of the phenyl ring of Phe329 was 9.0 Å versus
7.7 Å in the complex derived from the docking study. The distance
between the midpoint of the indole ring of Trp430 and the
midpoint of the phenyl ring of (+)-cocaine was 10.10 Å, whereas the
corresponding distance in the complex derived from the docking study
was 12.3 Å. In the MD-refined (+)-cocaine-BChE complex, the distance
between the ammonium nitrogen atom and the midpoint of the indole ring
of Trp82 was 5.7 Å; the distance between the ammonium
nitrogen atom and the side chain carbonyl carbon atom of
Glu197 was 4.2 Å; and the distance of the midpoint of the
phenyl ring of (+)-cocaine to the midpoint of the phenyl ring of
Tyr332 was 5.5 Å.
Experimental Support for the Models--
Despite a marked
difference in the experimentally observed kcat,
the difference in Km values between (+)- and
( Study of pH Dependence--
In both (
To test these predictions, the pH dependence of cocaine hydrolysis in a
series of enzyme kinetics experiments was examined (Fig.
5). As expected, the
kcat of (+)-cocaine was strongly
pH-dependent over the measured pH range of 5.5-8.5, with
the maximal kcat at pH 7.0 (Fig. 5). The
calculated pKa of 6.05 ± 0.04 for the
(+)-cocaine-BChE complex was derived from Equation 1. This result is
consistent with the catalytic triad theory for serine hydrolases,
namely that a neutral histidine (His438 in the present
case) is needed as a general base for enzymatic hydrolysis of
(+)-cocaine. In contrast, the kcat of
(
The same experimental data also yielded information on the pH
dependence of cocaine binding after the relationship of
kcat/Km versus pH
was analyzed according to Equation 2. The pKa values
for the free enzyme obtained from ( Explanation of Different Hydrolysis Rates--
The two predicted
Michaelis-Menten complexes reveal that the benzoic ester group of both
cocaine stereoisomers must rotate toward the catalytic
Ser198 for hydrolysis. However, in contrast to (+)-cocaine,
rotation of ( BChE Mutants for Hydrolysis of ( Data Deposition--
The coordinates of the two time-average
structures of (+)-cocaine-BChE and ( Conclusions--
We have predicted the Michaelis-Menten complexes
of BChE liganded with natural and unnatural cocaine molecules by
employing docking studies and MD simulations. In each complex, a
cocaine isomer binds in the catalytic site of BChE, with its ammonium group interacting with Trp82 via cation-pi interaction and
with its phenyl ring engaged in pi-pi interaction with
Tyr332. Both cocaine stereoisomers need to rotate their
benzoic ester group toward the catalytic Ser198 for
hydrolysis. In contrast to (+)-cocaine, Phe329 and
Trp430 are involved in pi-pi interactions with the phenyl
group of ()-cocaine only one-two
thousandth as fast as the unnatural (+)-stereoisomer. A starting point
in engineering BChE mutants that rapidly clear cocaine from the
bloodstream, for overdose treatment, is to elucidate structural factors
underlying the stereochemical difference in catalysis. Here, we report
two three-dimensional Michaelis-Menten complexes of BChE
liganded with natural and unnatural cocaine molecules, respectively,
that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both
cocaine stereoisomers must rotate toward the catalytic Ser198 for hydrolysis. Rotation of
(
)-cocaine appears to be hindered by interactions of its phenyl ring
with Phe329 and Trp430. These interactions do
not occur with (+)-cocaine. Because the rate of (
)-cocaine hydrolysis
is predicted to be determined mainly by the re-orientation step, it
should not be greatly influenced by pH. In fact, measured rates of this
reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can
explain why BChE hydrolyzes (+)-cocaine faster than (
)-cocaine, and
they suggest that mutations of certain residues in the catalytic site
could greatly improve catalytic efficiency and the potential for detoxication.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)-cocaine is only 3.9 min
1,
versus 33,900 min
1 for the optimal
substrate, butyrylthiocholine. We estimate that timely detoxication of
a cocaine overdose, with serum levels up to 20 mg/l (8), would require
at least 100 mg of enzyme. An A328Y mutant of BChE developed by the
trial and error approach (9) showed 4-5-fold improvement in
(
)-cocaine hydrolysis, but it is still not satisfactory for treatment
of cocaine overdose. Because human BChE hydrolyzes synthetic
(+)-cocaine (see Fig. 1) about 2,000-fold faster than the natural
(
)-cocaine (see Fig. 1) (9), elucidation of the structural factors
responsible for the different catalytic rates may offer a rational
strategy for engineering BChE mutants that can hydrolyze (
)-cocaine
rapidly enough to treat cocaine overdose.
)-cocaine via
docking and molecular dynamics (MD) simulation studies. We reasoned
that the overall hydrolysis rate constants (k3)
for both natural and unnatural cocaine molecules are relatively low as
compared with that for the good substrate, butyrylcholine. It was
therefore plausible to assume that the binding affinities of the
cocaine molecules would approximately reflect their
Km values. An advantage of this strategy was that
predictions of the Michaelis-Menten complexes avoided computationally
intensive ab initio calculations to address bond formation,
as would have been required in predicting transition state
enzyme-substrate complexes. In addition, Michaelis-Menten complexes
offer crucial information regarding the initial stage of substrate
binding that would not be available from the transition state
complexes. From this information, we could compare calculated intermolecular interaction energies and Km values. A correlation between experimental and calculated values would provide insights into the catalytic mechanisms for hydrolysis of cocaine isomers by BChE. The Michaelis-Menten complexes would also provide a
basis for predicting transition state complexes via ab
initio and potential of mean force calculations (19, 20), which in turn should help identify active site residues that hinder formation of
the transition state (
)-cocaine-BChE complex. Here, we report the following: 1) the predicted Michaelis-Menten complexes of BChE
liganded with (
)- and (+)-cocaine stereoisomers, respectively, 2)
correlation of the calculated intermolecular interaction energies of
such complexes with experimentally determined Km
values, and 3) pH dependence studies in support of the predicted
complexes and the structural factors responsible for different
hydrolysis rates of cocaine stereoisomers.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
anion was introduced next to the protonated, cationic residue if this
residue was more than 8 Å away from an anionic residue. His was
treated as protonated if it was less than 8 Å away from an acidic
residue, whereas the His438 residue, which was next to an
acidic residue but constituted the catalytic triad, was treated as
neutral. In the structure of the neutral His, one proton was attached
to the
nitrogen atom of the imidazole ring if the resulting
tautomer formed more hydrogen bonds in the protein; otherwise the
proton was attached to the
nitrogen atom. Cys was treated as
deprotonated when it formed a disulfide bond. The location of every
counter-ion was determined by an energy minimization, with a positional
constraint applied to all atoms of the system except for the
counter-ion. Such energy minimizations were performed with a nonbonded
cutoff of 8 Å and a dielectric constant of 1.0.
-H)
tautomer, whereas His372 was assigned as HIE
(N
-H); all the Glu and Asp residues were deprotonated
except that Glu441 was treated as neutral; and all the Arg
and Lys residues were deprotonated. Arg40,
Arg135, Arg138, Arg219,
Arg465, Arg470, Arg509,
Arg515, Lys9, Lys12,
Lys44, Lys51, Lys60,
Lys105, Lys131, Lys180,
Lys190, Lys248, Lys313,
Lys314, Lys458, Lys494,
Asp2, Asp70, Asp87,
Asp91, Asp268, Asp295,
Asp301, Asp324, Asp375,
Asp391, Asp454, Glu80,
Glu276, Glu308, Glu363, and
Glu404 were each neutralized by a nearby counter-ion
(Na+ or Cl
), respectively. In the MD
simulation, to neutralize the substrate-bound BChE structure, one
Na+ ion was added at the center of the mouth of the
catalytic gorge of BChE, and its final location was determined by
energy minimization. In the docking studies, the two Na+
ions added to neutralize Glu197 and Glu325 were
removed because they interfered with access of cationic substrate to
the active site.
)-cocaine molecules employing the CONSER
program (devised by Y.-P. Pang). This program first generated
conformations by specifying all discrete possibilities at 60° of arc
increment in a range of 0 to 360 for five rotatable torsions of cocaine
specified in Fig. 1. It then optimized
such conformers with the RESP charges and the Cornell et al.
(22) force field. Afterward, it performed a cluster analysis to delete
duplicates. These duplicates include those caused by C2 symmetry of the
phenyl ring. In the cluster analysis, two conformers were judged
different if at least one of the defined torsions differed by more than
30° of arc. The chiralities of the molecules were preserved during
energy minimizations by applying constraints on the chiral atoms and
their attached atoms.
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Fig. 1.
The natural (top) and
synthetic (bottom) cocaine structures and definitions
of five rotatable torsions used in the conformational
search.
, of 1.0 (IDIEL = 1.0); 4) the Berendsen
coupling algorithm (NTT = 1) (30); 5) the Particle Mesh
Ewald method (31) used to calculate the electrostatic interactions
(BOXX = 91.4869, BOXY = 77.6960, BOXZ = 69.3721, ALPHA = BETA = GAMMA = 90.0, NFFTX = 81, NFFTY = 81, NFFTZ = 64, SPLINE_ORDER = 4, ISCHARGED = 0, EXACT_EWALD = 0, DSUM_TOL = 0.00001); 6) the nonbonded atom
pair list updated at every 20 steps (NSNB = 20); 7) a
distance cutoff of 8.0 Å used to calculate the nonbonded steric
interaction (CUT = 8.0); and 8) defaults of other keywords not described here. The most energetically stable Michaelis-Menten complex of (
)-cocaine-BChE generated by the aforementioned docking study was used as the initial structure for the MD simulation. The
complex structure was simulated in a TIP3P (32) water box with a
periodic boundary condition at constant temperature (298 K) and
pressure (1 atm) for 1.0 ns (NCUBE = 20, QH = 0.4170, DISO = 2.20, DISH = 2.00, CUTX = CUTY = CUTZ = 8.2, NTB = 2, TEMPO = 298, PRESO = 1, TAUTP = 0.2, TAUTS = 0.2, TAUP = 0.2, NPSCAL = 0, and NTP = 1).
The solvated complex consisting of 50,941 atoms was first
energy-minimized for 500 steps to remove close van der Waals contacts
in the system. The minimized system was then slowly heated to 298 K (10 K/ps; NTX = 1) and equilibrated for 100 ps before a 1.0-ns
simulation. The time-average structure of 1000 instantaneous structures
of the complex at 1-ps intervals was generated by using the CARNAL
module of the AMBER 5.0 program.
)-cocaine was
measured by using a radiometric method developed in the Brimijoin group
to achieve adequate sensitivity. Details of assay performance and
validation will be published separately. Briefly,
[3H](
)-cocaine (222,000 dpm) diluted with varying
amounts of unlabeled (
)-cocaine was mixed with BChE in 0.1 M sodium phosphate buffer (pH 7.0) to a final volume of 100 µl. To get maximal signal, the reaction was incubated at 37 °C for
1 h. The enzymatic reaction was stopped by addition of 1 ml of
0.05 M HCl, and the product [3H]benzoic acid
was extracted with 4 ml of toluene for quantitation by scintillation
counting. Reaction with (+)-cocaine at 25 °C was simply monitored by
UV spectrophotometry (17), because modest amounts of BChE caused
readily measured hydrolysis. Vmax and
Km values were calculated by direct nonlinear
fitting to the Michaelis-Menten equation using Sigma Plot for Macintosh
(Jandel Scientific). To calculate the catalytic rate constant,
kcat, Vmax was divided by
the concentration of active sites, previously determined by titration
with echothiophate or diisopropyl fluorophosphate.
)-cocaine was measured by the
radiometric method described above, whereas that of (+)-cocaine was
measured by UV spectrophotometry at 240 nm. Spontaneous hydrolysis
rates in the absence of BChE (general base catalysis) were subtracted from the observed rates. Data were fitted to Equations 1 and 2.
(Eq. 1)
(Eq. 2)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)-cocaine
(derived from a conformational search) were docked to the active site of BChE by utilizing the EUDOC program. The docking results suggested that the ammonium nitrogen atom of (
)-cocaine interacts with Trp82 via cation-pi interaction in the most energetically
stable complex (Fig. 2). The distance
between the ammonium nitrogen atom and the midpoint of the indole ring
of Trp82 was 4.4 Å. The ammonium nitrogen atom of
(
)-cocaine also favorably interacted with the anionic residue
Glu197, in addition to the electrostatic interactions with
other anionic residues of the enzyme. The distance between the ammonium
nitrogen atom and the side chain carbonyl carbon atom of
Glu197 was 4.3 Å. The phenyl ring of (
)-cocaine engaged
in pi-pi interactions with Tyr332 and Phe329
(Fig. 2). The distances of the midpoint of the phenyl ring of (
)-cocaine to those of Tyr332 and
Phe329 were 5.0 and 5.8 Å, respectively. The
intermolecular interaction energy of this complex was
54.1 kcal/mol,
with a van der Waals contribution of
35.4 kcal/mol and an
electrostatic contribution of
18.7 kcal/mol (Table
I). The carbonyl carbon atom of the benzoic ester is 5.9 Å away from the oxygen atom of the catalytic Ser198, indicating that the benzoic ester group must rotate
toward Ser198 for hydrolysis. This rotation may
involve pivoting about the ammonium nitrogen atom that engaged in
cation-pi interaction with Trp82.
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Fig. 2.
Close-up view of the active sites of
the most energetically stable EUDOC-generated complexes of
( )-cocaine-BChE (top) and (+)-cocaine-BChE
(bottom). The perspective is looking down into
the active site of BChE. Hydrogen atoms are not displayed, for clarity.
The carbonyl carbon atoms and the hydroxyl oxygen atom are represented
with a ball model. The distances of the carbonyl carbon atom of the
methyl ester and of the benzoic ester to the hydroxyl oxygen atom of
Ser198 are 3.6 and 6.6 Å, respectively, in the
(+)-cocaine-BChE complex. The corresponding distances are 7.2 and 5.9 Å, respectively, in the (
)-cocaine-BChE complex.
Kinetic constants and relative intermolecular interaction energies of
human BChE for ()- and (+)-cocaine, respectively
)-cocaine.
)-cocaine to those of
Tyr332 and Phe329 were 5.0 and 5.8 Å,
respectively. Thus, these two aromatic residues of BChE have weaker
interactions with (+)-cocaine than with (
)-cocaine. The
intermolecular interaction energy of the (+)-cocaine-BChE complex was
about 2.0 kcal/mol higher than that of the complex with (
)-cocaine:
56.7 kcal/mol, with a van der Waals contribution of
30 kcal/mol and
an electrostatic contribution of
26.7 kcal/mol (Table I). The
carbonyl carbon atom of the benzoic ester is 6.6 Å away from the
oxygen atom of the catalytic Ser198, indicating that the
benzoic ester group must rotate toward Ser198 for
hydrolysis. This rotation may involve pivoting about the ammonium
nitrogen atom engaged in the cation-pi interaction with Trp82.
)- and
(+)-cocaine-BChE complexes were performed for the following reasons.
First, although the 3D structure of BChE used in this study had been
confirmed by site-directed mutagenesis studies (10), it had not been
tested with MD simulation in water. That kind of simulation was deemed
desirable to test self-consistency of the model. In a correct model,
there would be no large root mean square deviation between the initial
static and time-average structures (a tendency of unfolding) during the
MD simulation. A 1.0-ns MD simulation is certainly not long enough to
observe folding or unfolding of a protein. However, it is long enough to observe regional backbone conformational changes involving a partial
unfolding of a 3D structure that was incorrectly folded by homology
modeling. In other words, a "sustained" 1.0-ns MD simulation does
not necessarily validate a model, but a failed simulation calls for a
trial of alternatives (10-12). A second reason for MD simulation is
that no alternative conformations of BChE were used in the docking
studies to account for the molecular flexibility of BChE. MD
simulations of the cocaine-bound BChE complexes can help to convert the
most energetically stable conformation of BChE in the substrate-free
state to the most energetically stable conformation of BChE in the
substrate-bound state.
)-cocaine-BChE complex and the
corresponding time-average of 1,000 instantaneous structures saved at
1.0-ps intervals during the 1.0-ns MD simulation, we found a root mean square deviation of 1.4 Å (2,104 matched atoms) (Fig.
3). The corresponding root mean square
deviation for the (+)-cocaine-BChE complex was 1.5 Å (2,104 matched
atoms) (Fig. 3). The only regions that show significant differences
between the MD structure and the homology structure are L1
(Ile69-Gly78), L2
(Ala277-Thr284), L3
(Ile356-Ser362), and L4
(Tyr373-Arg381) on the protein surface (Fig.
3). These results suggest that the theoretical 3D model of BChE used in
the docking studies was reliable.
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Fig. 3.
Comparison of the static BChE
structure (red) with the corresponding time-averaged
structures (yellow) derived from 1000 instantaneous
structures of MD simulations of BChE liganded with ( )-cocaine
(top) and (+)-cocaine (bottom),
respectively. The root mean square deviations of the two overlaid
structures are 1.4 and 1.5 Å, respectively. The regions (L1,
Ile69-Gly78; L2,
Ala277-Thr284; L3,
Ile356-Ser362; and L4,
Tyr373-Arg381) that show significant
differences between the MD structure and the homology structure are
represented with a tube model.
)-cocaine-BChE complex derived
from the 1.0-ns MD simulation was consistent with the initial structure
derived from the docking study. The only difference between the two
structures was that the tilted T-shaped pi-pi interaction between the
phenyl ring of (
)-cocaine and Tyr332 in the
EUDOC-generated structure was changed to an off-center pi-pi
interaction in the time-averaged structure (Fig.
4). This conformational change was
promoted by an additional weak pi-pi interaction between the phenyl
ring of cocaine and Trp430, which was enabled by the
molecular flexibility of BChE during the MD simulation. The weak pi-pi
interaction was judged by the separation between the two aromatic
rings. The distance between the midpoint of the indole ring of
Trp430 and the midpoint of the phenyl ring of (
)-cocaine
was 6.3 Å, whereas the corresponding distance in the complex derived
from the docking study was 10.0 Å. In the (
)-cocaine-BChE complex refined by the 1.0-ns MD simulation, the distance between the ammonium
nitrogen atom and the midpoint of the indole ring of Trp82
was 4.7 Å; the distance between the ammonium nitrogen atom and the
side chain carbonyl carbon atom of Glu197 was 4.4 Å; and
the distances between the midpoint of the phenyl ring of (
)-cocaine
and the midpoints of the phenyl rings of Tyr332 and
Phe329 were 5.4 and 5.0 Å, respectively.
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Fig. 4.
Close-up view of the active sites of the
time-averaged ( )-cocaine-BChE (top) and
(+)-cocaine-BChE (bottom) complexes derived from
1.0-ns MD simulations (perspective looking down into the active site of
BChE). Hydrogen atoms are not displayed, for clarity. The carbonyl
carbon atoms and the hydroxyl oxygen atom are represented in a ball
model.
)-cocaine isomers was relatively small (Table I). For the poor
substrate, (
)-cocaine, Km can be used as a
measurement of the binding affinity of (
)-cocaine for BChE because of
the relatively small k3. For the better
substrate, (+)-cocaine, Km reflects but
underestimates the binding affinity of the ligand-enzyme complex. Actual Km values determined for the two cocaine
stereoisomers (Table I) indicate that the binding affinity of BChE for
(+)-cocaine is similar to that of (
)-cocaine but slightly higher.
This experimental result agrees with the theoretical prediction that
the intermolecular interaction energy between (+)-cocaine and BChE is
2.6 kcal/mol higher than that of (
)-cocaine, because the system error
of the EUDOC program is 0.5 kcal/mol.2 It thus supports the
two predicted cocaine-BChE complexes described above.
)- and (+)-cocaine-BChE
complexes, the carbonyl carbon atom of the benzoic ester is far away
from the hydroxyl oxygen atom of the catalytic Ser198 in
the active site of BChE, with distances of 5.9-6.1 Å for (
)-cocaine and 6.6-9.0 Å for (+)-cocaine. Therefore, both cocaine molecules need
to move their benzoic ester group close to the hydroxyl group of
Ser198 through rotation during the enzymatic reaction.
Furthermore, from inspection of the two complexes, it appears that the
rotation of (
)-cocaine is hindered by the strong interactions of its
phenyl ring with Tyr332, Phe329, and
Trp430, whereas the rotation of (+)-cocaine is hindered
only by the weak interaction of its phenyl ring with
Tyr332. A testable prediction is that hydrolysis of
(
)-cocaine would be less pH-dependent, because the
rate-determining step is the rotation of (
)-cocaine. In contrast, the
faster hydrolysis of (+)-cocaine would be strongly
pH-dependent, because the rate-determining step is the
formation of the acyl-enzyme intermediate.
)-cocaine was independent of pH over the measured pH range of
5.5-8.5 (Fig. 5). This result indicates that the rate-determining step
is the rotation of (
)-cocaine. In other words, the activation Gibbs energy of the enzymatic hydrolysis of (
)-cocaine is mainly determined by the free energy spent on pre-organization of the substrate. The
pH-dependent free energy required for acylation becomes
insignificant relative to the large free energy for pre-organization.
In the hydrolysis of (+)-cocaine, the pH-dependent free
energy required to form the acyl-enzyme intermediate is significant
relative to the small free energy spent on pre-organization. These
results therefore support our theoretical models revealing that the
phenyl ring of (
)-cocaine has stronger interactions with the active site of BChE than does that of (+)-cocaine.
View larger version (28K):
[in a new window]
Fig. 5.
Dependence of
kcat on pH, for (+)- and ( )-cocaine
stereoisomers. Apparent kcat values were
obtained by fitting substrate kinetics data to the Michaelis-Menten
equation (see "Materials and Methods"). The
kcat values, at 25 and 37 °C for (+)-and
(
)-cocaine isomers, respectively, were determined in 0.1 M sodium phosphate buffer at nine different pH values. For
hydrolysis of (+)-cocaine, kcat increased 5-fold
as pH rose from 5.5 to 8.5, whereas for hydrolysis of (
)-cocaine,
kcat was independent of pH over the same range
(note log scale on y axis).
)- and (+)-cocaine-BChE complexes
were 6.73 ± 0.14 and 6.77 ± 0.13, respectively. The two
nearly identical values are consistent with our theoretical models
indicating that these two substrates bind at nearly identical loci in
BChE. The results further suggest that the protonation state of the
water-accessible, catalytic His438 significantly affects
the binding of both cocaine isomers. As apparent from plots of
Km versus pH (Fig.
6), Km values of both
isomers indeed decrease as pH increases from 5.5 to 8.0. According to
the predicted 3D models of (
)- and (+)-cocaine-BChE complexes, the pH
dependence of both Km values is due to the repulsive
electrostatic interaction between the imidazonium group of
His438 substantially formed at pH < 6.73 ± 0.14 and the ammonium group of both cocaine isomers. However, the
Km of (
)-cocaine is somewhat more
pH-dependent than that of (+)-cocaine, as shown in Fig. 6,
because the k3 of (+)-cocaine is very low at
acidic pH but rises markedly in the pH range of 7.0-8.5, whereas the k3 of (
)-cocaine is low over the tested pH
range of 5.5-8.5.
View larger version (27K):
[in a new window]
Fig. 6.
Dependence of Km on
pH for both (+)- and ( )-cocaine. Experimental conditions were
the same as described in the legend to Fig. 5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)-cocaine appears to be hindered by pi-pi interactions
of its phenyl ring with the side chains of Phe329 and
Trp430. Consistent with this conclusion, experimental
studies showed that the hydrolysis of (+)-cocaine is strongly
pH-dependent, whereas the hydrolysis of (
)-cocaine is
pH-independent over the pH range from 5.5 to 8.5. These results
indicate that the activation Gibbs energy of the enzymatic hydrolysis
of (
)-cocaine is mainly determined by the free energy spent on
pre-organization of the substrate. The pH-dependent free
energy required for acylation becomes insignificant relative to the
large free energy for pre-organization. In contrast, this acylation
free energy significantly contributes to the activation Gibbs energy of
the enzymatic hydrolysis of (+)-cocaine. Therefore, the activation
Gibbs energy for (
)-cocaine must be greater than the activation Gibbs
energy for (+)-cocaine. The exponential relationship between the
hydrolysis rate constant and the activation Gibbs energy thus explains
why hydrolysis of (
)-cocaine is so much slower than that of
(+)-cocaine. The same relationship also indicates that pi-pi
interactions of the (
)-cocaine phenyl group with Phe329
and Trp430 contribute most to the larger activation energy
required for hydrolysis of this substrate. It is therefore conceivable
that Phe329 and Trp430 are the structural
factors responsible for the dramatic difference in hydrolysis of
different cocaine stereoisomers.
)-Cocaine--
The present study
suggests three rational strategies for engineering BChE mutants that
may rapidly hydrolyze (
)-cocaine. First, one could attempt moving the
catalytic triad to the vicinity of the benzoic ester of (
)-cocaine
(oriented in the Michaelis-Menten complex predicted by the present
study). Such a change should allow the substrate to be hydrolyzed more
rapidly by enzyme catalysis per se. Before attempting such
experiments, however, extensive computational studies are needed to
ensure that a mutant BChE with relocated catalytic triad would bind
cocaine in the same way as would the wild type enzyme. A second
strategy is to mutate residues in the catalytic site to disable the
primary binding site of (
)-cocaine in the hope that an alternative
binding site would position the benzoic ester close to
Ser198. Third, one could delete the bulky side chains that
appear likely to hinder the movement of the benzoic ester toward
Ser198. Candidates are Tyr332,
Phe329, and Trp430, which appear to stabilize
the ground state binding orientation of (
)-cocaine, and
Phe329, Phe398, and Leu286, which
may sterically hinder the movement of the benzoic ester along the path
predicted by visual inspection. Using the hypothetical Michaelis-Menten
complexes developed in this study, a prediction of the transition state
complexes via ab initio calculations and potential of mean
force calculations (19, 20) may shed additional light on this potential approach.
)-cocaine-BChE derived from
1.0-ns (1.0 fs time step) MD simulations were deposited in the Protein
Data Bank (27, 33) on February 22, 2000 (Protein Data Bank codes 1EHQ
and 1EHO). Residue numbers in the structures deposited at the PDB deviate by one from the residue numbers reported in the literature and
in this article.
)-cocaine, therefore greatly hindering the rotation of the
benzoic ester group of (
)-cocaine. On the basis of the two 3D models,
we predicted that rotation of (+)-cocaine would be rapid and that the
rate-limiting step in hydrolysis would involve acylation. Thus
hydrolysis of (+)-cocaine would be expected to be heavily influenced by
pH. We predicted further that rotation of (
)-cocaine would be
hindered by pi-pi interaction involving its phenyl ring and that the
rate-limiting step in hydrolysis would be determined mainly by the rate
of substrate reorientation. Thus hydrolysis of (
)-cocaine would be
less pH-sensitive. Experimental studies confirmed both of these
predictions. The two 3D cocaine-BChE complexes offer an explanation of
why BChE hydrolyzes (+)-cocaine much faster than (
)-cocaine,
suggesting that mutations of several residues in the catalytic site may
lead to BChE mutants that hydrolyze natural cocaine rapidly enough for
clinical use in treating cocaine overdose.
![]() |
ACKNOWLEDGEMENT |
---|
We thank J. L. Sussman of the Weizmann Institute of Science for providing the coordinates of the theoretical structure of human BChE.
![]() |
FOOTNOTES |
---|
* This work was supported by the Mayo Foundation for Medical Education and Research and National Institutes of Health Grant DA011707 (to O. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Tables I and II.
To whom correspondence should be addressed. E-mail:
pang@mayo.edu.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M006676200
2 Y. P. Pang, E. Perola, K. Xu, and F. G. Prendergast, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BChE, butyrylcholinesterase; 3D, three-dimensional; MD, molecular dynamics.
![]() |
REFERENCES |
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