Introduction

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Cocaine overdose remains a serious problem with no broadly effective remedies (Wetli, 1987; Schrank, 1992; Hollander, 1995; Marzuk et al., 1995). An alternative to conventional therapy with receptor antagonists is to enhance metabolic inactivation (Landry et al., 1993; Gorelick, 1997). Cocaine metabolism is partly driven by carboxylesterase. For detoxication, however, the more relevant enzyme is butyrylcholinesterase (BChE), which converts cocaine to the less active derivatives ecgonine methyl ester and benzoic acid (Inaba et al., 1978). Pretreating rats with human BChE reduces cocaine-induced cardiac effects (Mattes et al., 1996) and can prevent death (Lynch et al., 1997), but BChE has such low efficiency that huge amounts could be needed to detoxify a human patient. Clearly, practical treatment requires a cocaine hydrolase with improved kinetic properties.

Because human BChE hydrolyzes unnatural (+)-cocaine 2000-fold faster than naturally occurring ()-cocaine, we recently used molecular modeling to investigate BChE complexes with each of these stereoisomers (Sun et al., 2001). Our model predicted that both forms of cocaine bind similarly within the active site of BChE but not oriented for immediate catalysis. We hypothesized that the differing catalytic efficiencies with these two substrates reflect differences in a key step: rotation of cocaine's benzoic ester group toward the catalytic triad. Inspection of the model suggested that (+)-cocaine could easily rotate about the axis formed by a cation- interaction between its ammonium group and Trp82 of BChE. In contrast, ()-cocaine would rotate less readily because of steric hindrance and a relatively strong - interaction with Tyr332.

Accordingly, we developed a rational strategy to create an effective cocaine hydrolase from BChE in two steps: 1) replacing Tyr332 with Ala, to reduce the steric hindrance and the - interaction that impede rotation and 2) replacing Ala328 with Trp to provide a cation- interaction (Gallivan and Dougherty, 2000) to restore substrate affinity lost in disabling the - interaction. Herein, we report the properties and some applications of this re-engineered BChE, along with further molecular modeling studies aimed at explaining its improved catalytic efficiency with cocaine.

	Experimental Procedures

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Materials. Reagents for producing recombinant BChE included a site-directed mutagenesis kit (Stratagene, La Jolla, CA), an expression plasmid pRc/CMV incorporating human BChE (Xie et al., 1999), Dulbecco's modified Eagle's medium (DMEM; Fisher Scientific, Fairlawn, NJ), human embryonic kidney 293 cells (American Type Culture Collection, Manassas VA), and a plasmid purification kit (QIAGEN, Valencia, CA). Oligonucleotides were synthesized by Mayo's Molecular Biology Core Facility. Echothiophate iodide was from Wyeth-Ayerst (Rouses Point, NY). Synthetic (+)-cocaine was provided by the National Institute on Drug Abuse Research Resources Drug Supply System (Rockville, MD), and ()-[³H]cocaine was purchased from New England Nuclear (Boston, MA). Other materials, all from Sigma (St. Louis, MO) were: natural ()-cocaine (purchased under an institutional license); butyrylthiocholine iodide; benzoylcholine chloride; and human plasma.

Molecular Modeling. The 3D structure of A328W/Y332A was generated from the computationally generated 3D model of wild-type BChE by changing residues A328 and Y332 to tryptophan and alanine, respectively, with the LINK, EDIT, PARM, and SANDER modules of the AMBER 5.0 program (Pearlman et al., 1995). Cocaine was docked to the catalytic gorge of A328W/Y332A by the EUDOC program (ceramic version) as described previously (Pang et al., 2001). A box of 5.5 × 4.0 × 10.0 Å was defined in the binding pocket of A328W/Y332A to confine ligand translation. Translational and rotational increments were set at 1.0 Å and 10° of arc, respectively. The most energetically stable, EUDOC-generated, Michaelis-Menten complex of cocaine and enzyme was then refined by a molecular dynamic simulation. Complexes were simulated for 1.0 ns in a TIP3P water box with a periodic boundary condition at constant temperature (298°K) and pressure (1 atm). A time-average of 1000 instantaneous structures of the complex at 1-ps intervals was generated with the CARNAL module of AMBER 5.0.

Mutagenesis of BChE. Mutations were generated from wild-type human BChE in a pRc/CMV expression plasmid. Using plasmid DNA as template and primers with specific base-pair alterations, mutations were made by polymerase chain reaction with Pfu polymerase, for replication fidelity. Modified plasmid DNA was transformed into Escherichia coli, amplified, and re-sequenced to ensure correctness. Purified plasmids were then transfected into human embryonic kidney 293 cells by calcium phosphate precipitation, and recombinant BChE was expressed in serum-free DMEM.

Expression and Purification of BChE Tetramers. Stable lines expressing human BChE were made in CHO-K1 cells (American Type Culture Collection). As described previously (Xie et al., 1999), a 1.8-kilobase DNA sequence encoding the signal peptide and 574 amino acids of the enzyme was cloned into pGS. To promote BChE assembly into a stable tetrameric form, this plasmid was cotransfected with a pRc/RSV plasmid (Invitrogen, Carlsbad, CA) that expressed the signal peptide and 45 N-terminal amino acids of the rat COLQ gene (Krejci et al., 1997; Altamirano and Lockridge, 1999). Colonies resistant to 50 µM methionine sulfoximine and 0.8 mg/ml G418 were expanded as 1-liter roller cultures (Ultraculture medium plus methionine sulfoximine and G418 alternated with glutamine-deficient DMEM/Ham's F-12). The secreted BChE was purified by affinity chromatography on procainamide-Sepharose followed by anion exchange chromatography on DE52 (Arpagaus et al., 1990). Purified BChE was dialyzed in phosphate-buffered saline, concentrated to 1 mg/ml, filter sterilized, and stored at 4°C. Active sites in the preparation were titrated by overnight incubation at 25°C with echothiophate (Radic et al., 1991).

Enzyme Assays and Kinetics. To measure hydrolysis of efficient substrates butyrylcholine and synthetic (+)-cocaine, classical spectrophotometric methods were used (Ellman et al., 1961; Gatley, 1991). To measure the slower hydrolysis of natural cocaine we devised a sensitive method based on liberation of [³H]benzoic acid from ()-[³H]cocaine. The radiolabeled substrate was pre-extracted with toluene to eliminate any free benzoic acid, then mixed with unlabeled cocaine. Substrate mixtures (50 nCi, 50 µl) were incubated for 60 min in scintillation vials with 50 µl of enzyme in 0.1 M sodium phosphate, pH 7.4. Reactions were stopped by addition of 300 µl of 0.02 M HCl to neutralize liberated benzoic acid. After partitioning into 4 ml of toluene-based fluor (or, with high protein samples, after extraction into pure toluene), product was measured by scintillation counting. In some cases residual substrate was measured identically with one exception: before extraction, samples were alkalinized with 300 µl of 1 M Na₂CO₃, so neutral [³H]cocaine (but not ionized benzoic acid) would partition into the organic phase. To obtain a signal 2-fold above background in the radiometric assay, only 0.001 unit of wild-type BChE was required, about 10,000 less than typically used in spectrophotometric assays for cocaine hydrolysis. However, the two methods yielded similar estimates of K_M and V_max.

Tissue and Plasma Samples. To simulate cocaine clearance in vitro, 1-ml samples at pH 7.6 were prepared with 0.85 ml of human plasma plus 1) phosphate-buffered saline, 2) wild-type or mutant BChE (4.2 µg/ml), or 3) BChE and inhibitor (echothiophate, 0.1 mM). For in vivo studies, male Sprague-Dawley rats (250~350 g) were anesthetized with urethane (1.45 mg/kg, i.p.). Catheters were placed in the tail vein and carotid artery of each rat, and A328W/Y332A was administered (1 or 3 mg/kg, i.v.), followed 10 min later by ()-[³H]cocaine (6.8 mg/kg, 30 µCi, total). Fifteen minutes later, 1 ml of arterial blood was drawn into a heparinized tube. The rats were then euthanized with sodium pentobarbital (200 mg/kg, i.v.) and perfused with 60 ml of NaCl solution containing inhibitors of BChE and carboxylesterase (echothiophate, 10⁵M; tetraisopropyl pyrophosphoramide, 10⁵M; and saturated sodium fluoride, 25 µl/ml). Brain, heart, kidney, liver, and spleen were collected on dry ice, then homogenized in cold 10 mM sodium phosphate, pH 7.4, with 0.5% Tween 100 and enzyme inhibitors. After centrifugation (8000g, 10 min, 4°C), the supernatants were immediately assayed for cocaine.

Locomotor Activity. Adult male 129Sv mice (52-94 days old, 24-35 g), kept in cages without bedding that could spuriously trigger a beam counter, were acclimated for 1 h in a dimly lit, sound-proof room. Mice then received either cocaine alone (25 mg/kg, i.p.), saline alone, or BChE (2.8 IU/g) followed 1 h later by cocaine. Each mouse received cocaine only once. Motion was detected as beam interruptions with an opposed light-emitting diode and a photodiode detector connected to a microprocessor for quantitation.

Statistical Analysis. Treatment effects were subjected to analysis of variance using StatView 4.5 (Abacus Concepts, Berkeley, CA); p < 0.05 was considered statistically significant.

	Results

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Improving Cocaine Hydrolase Activity. Modified BChE with double mutations predicted to enhance cocaine hydrolase activity (A328W/Y332A) was stably expressed and purified in milligram quantities. Compared with wild-type BChE, the two mutations synergistically caused a 40-fold increase in catalytic power (k_cat) versus ()-cocaine and an 11-fold rise in catalytic efficiency (k_cat /K_M), with little apparent decline in affinity (Table 1). By contrast, k_cat /K_M with butyrylthiocholine was nearly the same in the mutant (580 ± 12 min¹µM¹) as in wild-type BChE (595 ± 2.6 min¹µM¹), whereas k_cat /K_M for (+)-cocaine actually fell almost 2-fold (from 760 ± 3 to 460 ± 16 min¹µM¹). Hence, the selected mutations selectively enhanced hydrolysis of clinically relevant ()-cocaine. It was also striking that k_cat with both cocaine stereoisomers was pH-dependent in A328W/Y332A, whereas that of wild-type BChE was pH-dependent only with the unnatural isomer (Fig. 1).

View this table: [in this window] [in a new window]   TABLE 1 Kinetic constants of BCHE and mutants with ()-cocaine Hydrolysis of ()-cocaine was determined radiometrically. Data were fitted to the Michaelis-Menten equation by nonlinear regression; Vmax was divided by titrated enzyme concentrations to calculate kcat.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Cocaine hydrolysis and pH-dependence. Reactions were carried out in triplicate in 0.1 M sodium phosphate buffers at 25°C for (+)-cocaine () and 37°C for ()-cocaine (). Apparent kcat values (min1) were determined after fitting kinetic data directly to the Michaelis-Menten equation.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Cocaine complexes with A328W/Y332A and wild-type BChE. Molecular modeling was performed according to protocols described previously (Sun et al., 2001). Close-up views (looking down into active site) of time-averaged complexes from 1.0 ns (1.0-fs time step) molecular dynamics simulations. Top, ()-cocaine-BChE [protein data bank (PDB) code: 1EHO); middle, ()-cocaine-A328W/Y332A (PDB code: 1KCJ); bottom, (+)-cocaine-BChE (PDB code: 1EHQ).

Enhancing Cocaine Metabolism. A rationale to use hydrolases for cocaine detoxication is that circulating enzymes should draw drug from target tissues by reversing concentration gradients. With its 40-fold increase in k_cat for ()-cocaine and its K_M in the concentration range typical of fatal overdose (Wetli, 1987), A328W/Y332A should be more clinically effective than wild-type BChE. This prediction was tested first on cocaine removal from human plasma in vitro (Fig. 3). Cocaine half-life in normal plasma was 154 min. Adding wild-type BChE (4.2 µg/ml) reduced half-life to 86 min, but the same amount of A328W/Y332A reduced half-life to 5 min and converted all cocaine to benzoic acid within 30 min. A328W/Y332A was also highly effective in vivo. When injected into rats before cocaine challenge, it caused dose-dependent reduction of cocaine levels in all sampled tissues, including heart and brain (Fig. 4). Enzyme inactivated by echothiophate did not alter cocaine removal (not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Accelerated cocaine breakdown in plasma. Cocaine breakdown was measured in vitro in human plasma plus wild-type BChE or A328W/Y332A (4.2 µg/ml) in a final volume of 1 ml (pH 7.4-7.7). After addition of [3H]cocaine, separate aliquots were sampled periodically to assay residual [3H]cocaine (top) and liberated [3H]benzoic acid (bottom).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   A328W/Y332A pretreatment and tissue cocaine levels. A328W/Y332A was injected 10 min before [3H]cocaine, and cocaine levels were assayed in tissue samples collected 15 min later. Data are means ± S.E.M. in micrograms per gram, wet weight (n = 5). , significantly different from control samples by analysis of variance and post hoc Scheffé's F test (p < 0.05). Regression analysis demonstrated dose-dependent, A328W/Y332A-induced reduction of cocaine levels in all tissues including brain (p < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Locomotor activity in mice as a measure of cocaine toxicity. Mice were pretreated with saline or BChE (wild-type or mutant, 2.8 units/g, i.p.) 1 h before receiving cocaine (25 mg/kg, i.p.). Beam breaks were summed every 2 min for 60 min after injection of cocaine (mean values are shown, n = 10). The loss of cocaine-induced hyperactivity in mice treated with A328W/Y332A BChE was highly significant (p < 0.001).

	Discussion

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Why A328W/Y332A Mutations Improve Cocaine Hydrolysis. Although cocaine's two isomers differ more than 1000-fold in rates of hydrolysis, wild-type BChE binds them nearly equally well (Gatley, 1991; Berkman et al., 1997; Xie et al., 1999; Sun et al., 2001). We argue that BChE's low efficiency with ()-cocaine is caused not by weak binding or poor catalysis per se but by difficulty in moving substrate from binding site to active site. The assembled data on pH-dependence support the view that substrate movement is important for cocaine hydrolysis by BChE. Our initial studies with native BChE had shown a pH-dependent k_cat for hydrolysis of (+)-cocaine, but a pH-independent k_cat for hydrolysis of the poor substrate, ()-cocaine (Sun et al., 2001). The implication was that formation/decay of the acyl-enzyme intermediate is the rate-limiting step with (+)-cocaine, whereas the rate-limiting step with ()-cocaine occurs earlier. More than one interpretation can be considered, but we lean toward the view that, after initial binding, ()-cocaine is selectively hindered in rotating into close contact with residues of the catalytic triad. Because the differential effects of pH are lost in the double mutant enzyme, we suggest that this hindrance has been reduced or eliminated in A328W/Y332A.

Clinical Potential of A328W/Y332A. It has been estimated that a cocaine hydrolase with k_cat >120 min¹ and K_M < 30 µM would be effective against overdose (Landry et al., 1993). A328W/Y332A meets these criteria. In fact, the enzymatic properties of A328W/Y332A, despite its modestly elevated K_M, give this enzyme a considerable advantage in eliminating cocaine from the blood circulation, especially when plasma levels are high. Patients intoxicated with cocaine can have drug levels up to 60 µM (Wetli, 1987; Benowitz, 1993), a concentration that just reaches the saturation point for A328W/Y332A.