Predicted Michaelis-Menten Complexes of Cocaine-Butyrylcholinesterase

ENGINEERING EFFECTIVE BUTYRYLCHOLINESTERASE MUTANTS FOR COCAINE DETOXICATION*,

Hong SunDagger §, Jamal El Yazal§, Oksana Lockridge, Lawrence M. Schopfer, Stephen BrimijoinDagger §, and Yuan-Ping PangDagger §||**DaggerDagger

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (-)-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.

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 (-)-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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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- 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 delta  nitrogen atom of the imidazole ring if the resulting tautomer formed more hydrogen bonds in the protein; otherwise the proton was attached to the epsilon  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.

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 (Ndelta -H) tautomer, whereas His372 was assigned as HIE (Nepsilon -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.

Conformational Searches-- Conformational searches were performed for (+)- and (-)-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.

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, epsilon , 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.

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 (-)-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.

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 (-)-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.


k<SUB><UP>cat app</UP></SUB>=<FR><NU>k<SUB><UP>cat</UP></SUB></NU><DE>1+<FR><NU>[<UP>H</UP><SUP><UP>+</UP></SUP>]</NU><DE>K<SUB>a</SUB></DE></FR></DE></FR> (Eq. 1)

(k<SUB><UP>cat</UP></SUB>/K<SUB>m</SUB>)<SUB><UP>app</UP></SUB>=<FR><NU>k<SUB><UP>cat</UP></SUB>/K<SUB>m</SUB></NU><DE>1+<FR><NU>[<UP>H</UP><SUP><UP>+2</UP></SUP>]</NU><DE>K<SUB>a</SUB></DE></FR></DE></FR> (Eq. 2)

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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cocaine-BChE Complexes Derived from Docking Studies-- Altogether, 15 different conformations of (-)-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.

                              
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Table I
Kinetic constants and relative intermolecular interaction energies of human BChE for (-)- and (+)-cocaine, respectively
E, total interaction energy; Evdw, interaction energy contributed by van der Waals interactions; Eele, interaction energy contributed by electrostatic interactions. Km and kcat values were derived from UV spectrophotometric observations of reactions run in pH 7.0 sodium phosphate buffer, at 25 °C for (+)-cocaine and 37 °C for (-)-cocaine.

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 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.

Cocaine-BChE Complexes Refined by MD Simulations-- Separate 1.0-ns (1.0 fs time step) MD simulations of the (-)- 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.

In the present case, comparing all non-hydrogen, protein backbone atoms in the initial (-)-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.

The time-averaged structure of the (-)-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.

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 (-)-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.

Study of pH Dependence-- In both (-)- 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.

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 (-)-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.


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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).

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 (-)- 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.


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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

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 (-)-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.

BChE Mutants for Hydrolysis of (-)-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.

Data Deposition-- The coordinates of the two time-average structures of (+)-cocaine-BChE and (-)-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.

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, 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.

Dagger Dagger 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.

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