Predicted Michaelis-Menten Complexes of Cocaine-Butyrylcholinesterase

Abstract

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 Ser 198 for hydrolysis. Rotation of (؊)-cocaine appears to be hindered by interactions of its phenyl ring with Phe 329 and Trp 430 . 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.Cocaine overdose is a leading cause of death among urbandwelling 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 k cat of BChE for (Ϫ)-cocaine is only 3.9 min Ϫ1, versus 33,900 min Ϫ1 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 (Ϫ)-cocaine hydrolysis, but it is still not satisfactory for treatment of cocaine overdose. Because human BChE hydrolyzes synthetic (ϩ)-cocaine (see Fig. 1) about 2,000-fold faster than the natural (Ϫ)-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 (Ϫ)-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 o...

Free Energy Perturbation (FEP) Simulation on the Transition States of Cocaine Hydrolysis Catalyzed by Human Butyrylcholinesterase and Its Mutants

“…23 A328W/Y332A BChE has a ∼9.4-fold improved catalytic efficiency (k cat /K M ) compared to wild-type BChE against (-)-cocaine (k cat = 4.1 min -1 and K M = 4.5 μM). 23 The catalytic efficiency (k cat /K M ) of A328W/Y332G BChE was estimated to be slightly higher than k cat /K M of A328W/Y332A BChE against (-)-cocaine in our previous work (although the specific k cat and K M values were not determined yet), 24 which is qualitatively consistent with the ΔΔG(A328W/Y332A→A328W/Y332G) value determined by the FEP simulations. The experimental catalytic efficiency of A328W/Y332G/A199S BChE has not been reported previously in literature.…”

“…21,22 Some mutants of human BChE reported in literature have an improved catalytic efficiency (k cat /K M ) against (-)-cocaine. 23,24,25,26 It is still highly desirable for development of a more efficient anti-cocaine medication to design and discover BChE mutants with a significantly improved catalytic efficiency against (-)-cocaine. A high-activity mutant of human BChE is expected to serve as an exogenous enzyme for injection so as to accelerate (-)-cocaine hydrolysis before (-)-cocaine crossing BBB to reach CNS.…”

“…Further computational modeling also suggests that the formation of the prereactive BChE-(-)-cocaine complex (ES) is hindered mainly by the bulky side chain of Y332 residue in wild-type BChE, but the hindering can be removed by the Y332A or Y332G mutation. 24 Therefore, starting from the A328W/Y332A 23 or A328W/ Y332G 24 mutant, the rational design of further mutation(s) to improve the catalytic efficiency of BChE against (-)-cocaine can aim to decrease the free energy barrier for the first reaction step without significantly affecting the ES formation and other chemical reaction steps. The analysis of previous experimental and computational data available in literature 19,23,27 clearly shows that the rate-determining step of (-)-cocaine hydrolysis catalyzed by the A328W/Y332A mutant is the first step of the chemical reaction process.…”

“…24 Therefore, starting from the A328W/Y332A 23 or A328W/ Y332G 24 mutant, the rational design of further mutation(s) to improve the catalytic efficiency of BChE against (-)-cocaine can aim to decrease the free energy barrier for the first reaction step without significantly affecting the ES formation and other chemical reaction steps. The analysis of previous experimental and computational data available in literature 19,23,27 clearly shows that the rate-determining step of (-)-cocaine hydrolysis catalyzed by the A328W/Y332A mutant is the first step of the chemical reaction process. Thus, the modeling of the transitional state of this first reaction step (TS1, see Scheme 1) is important for rational design of highactivity mutants of BChE.…”

Rational design of an enzyme mutant for anti-cocaine therapeutics

“…The MD trajectories for the non-prereactive BChE-(−) cocaine and BChE-(+)-cocaine complexes are also very stable. In addition, the simulated nonprereactive BChE-(−)-cocaine and BChE-(+)-cocaine complexes are very close to the simulated Michaelis-Menten complexes reported by Sun et al [38]. All these suggest that the binding of BChE with (−)-cocaine and (+)-cocaine is similar to those proposed with butyrylthiocholine and succinyldithiocholine.…”