Catalytic mechanism and energy barriers for butyrylcholinesterase-catalyzed hydrolysis of cocaine

Abstract

The geometries of the transition states, intermediates, and prereactive enzyme-substrate complex and the corresponding energy barriers have been determined by performing hybrid quantum mechanical/molecular mechanical (QM/MM) calculations on butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)- and (+)-cocaine. The energy barriers were evaluated by performing QM/MM calculations with the QM method at the MP2/6-31+G* level and the MM method using the AMBER force field. These calculations allow us to account for the protein environmental effects on the transition states and energy barriers of these enzymatic reactions, showing remarkable effects of the protein environment on intermolecular hydrogen bonding (with an oxyanion hole), which is crucial for the transition state stabilization and, therefore, on the energy barriers. The calculated energy barriers are consistent with available experimental kinetic data. The highest barrier calculated for BChE-catalyzed hydrolysis of (-)- and (+)-cocaine is associated with the third reaction step, but the energy barrier calculated for the first step is close to the highest and is so sensitive to the protein environment that the first reaction step can be rate determining for (-)-cocaine hydrolysis catalyzed by a BChE mutant. The computational results provide valuable insights into future design of BChE mutants with a higher catalytic activity for (-)-cocaine.

FIGURE 1

BChE-catalyzed hydrolysis of (−)- and (+)-cocaine.

FIGURE 2

Schematic representation of the pathway for BChE-catalyzed hydrolysis of (−)-cocaine; the pathway for (+)-cocaine is the same in terms of the covalent bond formation and breaking, as the only difference between (−)-cocaine and (+)-cocaine is the position of the methyl ester group. Only the QM-treated high-layer part of the reaction system in the ONIOM (QM/MM) calculations are drawn. Notation [H] refers to a nonhydrogen atom in the MM-treated low-layer part of the protein, and the cut covalent bond with this atom is saturated by a hydrogen atom. The dashed lines in the transition state structures represent the covalent bonds that form or break during the reaction steps.

FIGURE 3

Part of the QM/MM-optimized geometry of the transition state for the first step (TS1) of (−)-cocaine hydrolysis catalyzed by the wild-type BChE. The atoms highlighted as balls include several key H atoms (gray balls) and the C and O atoms in the carboxylate group of cocaine (yellow balls).

FIGURE 4

Part of the QM/MM-optimized geometry of the transition state for the second step (TS2) of (−)-cocaine hydrolysis catalyzed by the wild-type BChE. The atoms highlighted as balls include several key H atoms (gray balls) and the C and O atoms in the carboxylate group of cocaine (yellow balls).

FIGURE 5

Part of the QM/MM-optimized geometry of the transition state for the third step (TS3) of (−)/(+)-cocaine hydrolysis catalyzed by the wild-type BChE. The atoms highlighted as balls include several key H atoms (gray balls) and the C and O atoms in the carbonyl group of cocaine (yellow balls).

FIGURE 6

Part of the QM/MM-optimized geometry of the transition state for the fourth step (TS4) of (−)/(+)-cocaine hydrolysis catalyzed by the wild-type BChE. The atoms highlighted as balls include several key H atoms (gray balls) and the C and O atoms in the carbonyl group of cocaine (yellow balls).

FIGURE 7

Part of the QM/MM-optimized geometry of the transition state for the first step (TS1) of (+)-cocaine hydrolysis catalyzed by the wild-type BChE. The atoms highlighted as balls include several key H atoms (gray balls) and the C and O atoms in the carboxylate group of cocaine (yellow balls).

FIGURE 8

Part of the QM/MM-optimized geometry of the transition state for the second step (TS2) of (+)-cocaine hydrolysis catalyzed by the wild-type BChE. The atoms highlighted as balls include several key H atoms (gray balls) and the C and O atoms in the carboxylate group of cocaine (yellow balls).

FIGURE 9

Plots of the key internuclear distances (in angstroms) versus the time in the simulated TS1 structure for (−)-cocaine hydrolysis catalyzed by A199S/A328W BChE. Traces D1, D2, and D3 refer to the distances between the carbonyl oxygen of (−)-cocaine and the NH hydrogen of G116, G117, and S199, respectively. Trace D4 is the internuclear distance between the carbonyl oxygen of (−)-cocaine and the hydroxyl hydrogen of the S199 side chain in A199S/A328W BChE. RMSD represents the root mean-square deviation (in angstroms) of the simulated positions of the protein backbone atoms from those in the initial structure.