Fundamental Reaction Mechanism for Cocaine Hydrolysis in Human Butyrylcholinesterase

Abstract

Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.

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

“…35 The geometries used in the ESP calculations came from those obtained from previous ab initio reaction coordinate calculations. 19 The charges of the atoms of the oxyanion hole and catalytic triad remain the same with those available in Amber7 package.…”

“…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.…”

“…4,18 First, the metabolites of cocaine catalyzed by this enzyme are all biologically inactive and, therefore, the metabolites produced in this pathway have no toxic effects as cocaine. 19 Second, human BChE has a long history of clinic use, and no adverse effect has been noted. Finally, about 20 different naturally occurring mutants of human BChE have been identified, and there is no evidence that these mutants are antigenic.…”

Structure-and-mechanism-based design and discovery of therapeutics for cocaine overdose and addiction

“…41 , 42 , 43 , 44 ,45 This mechanistic hypothesis has been supported by reported reaction coordinate calculations using the first-principles quantum mechanics (QM) and hybrid quantum mechanics and molecular mechanics (QM/ MM) methods. 38,46 Based on the QM and QM/MM reaction coordinate calculations, the first step of the chemical reaction process is initialized by S198 O γ attack at the carbonyl carbon of the cocaine benzoyl ester to form the first tetrahedral intermediate (INT1) through the first transition state (TS1). Fig.…”

“…A detailed analysis38 , 47 of the MD-simulated structures of wild-type BChE binding with (−)-cocaine and (+)-cocaine revealed that Y332 is a key residue hindering the structural change from the non-prereactive BChE-(−)-cocaine complex to the prereactive BChE-(−)-cocaine complex.38 , 47 A number of possible mutants of BChE were proposed for in vitro experimental tests. 38,47,48, 49 , 50 , 51 ,52 The earliest design of BChE mutants was only based on the modeled or simulated structure of the non-prereactive BChE-(−)-cocaine complex with wild-type BChE; the possible dynamics of the proposed BChE mutants were not examined. Some of the proposed mutants indeed have a significantly improved catalytic efficiency against (−)-cocaine, 38, 47 -52 e.g.…”