Reaction pathway and free energy profiles for butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine

Abstract

The catalytic mechanism for butyrylcholineserase (BChE)-catalyzed hydrolysis of acetylthiocholine (ATCh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy (QM/MM-FE) calculations on both acylation and deacylation of BChE. Additional quantum mechanical (QM) calculations have been carried out, along with the QM/MM-FE calculations, to understand the known substrate activation effect on the enzymatic hydrolysis of ATCh. It has been shown that the acylation of BChE with ATCh consists of two reaction steps including the nucleophilic attack on the carbonyl carbon of ATCh and the dissociation of thiocholine ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of substrate and dissociation between the carboxyl carbon of substrate and hydroxyl oxygen of Ser198 side chain. QM/MM-FE calculation results reveal that the acylation of BChE is rate-determining. It has also been demonstrated that an additional substrate molecule binding to the peripheral anionic site (PAS) of BChE is responsible for the substrate activation effect. In the presence of this additional substrate molecule at PAS, the calculated free energy barrier for the acylation stage (rate-determining step) is decreased by ~1.7 kcal/mol. All of our computational predictions are consistent with available experimental kinetic data. The overall free energy barriers calculated for BChE-catalyzed hydrolysis of ATCh at regular hydrolysis phase and substrate activation phase are ~13.6 and ~11.9 kcal/mol, respectively, which are in reasonable agreement with the corresponding experimentally derived activation free energies of 14.0 kcal/mol (for regular hydrolysis phase) and 13.5 kcal/mol (for substrate activation phase).

Figure 1

Key states for the acylation reaction stage of BChE-catalyzed ATCh hydrolysis. The geometries were optimized at the QM/MM(B3LYP/6-31+G*:AMBER) level. The key distances in the figure are in Å. Carbon, oxygen, nitrogen, sulfur, and hydrogen atoms are colored in green, red, blue, yellow, and white, respectively. The backbone of the protein is rendered in orange. The QM atoms are represented as balls and sticks and the surrounding residues are rendered as sticks or lines. The figures below are represented using the same method.

Figure 2

Key states for the deacylation reaction of BChE-catalyzed ATCh hydrolysis. The geometries were optimized at QM/MM(B3LYP/6-31+G*:AMBER) level. See caption of Figure 1 for the color codes for different types of atoms.

Figure 3

Free energy profiles for the acylation and deacylation stages of BChE-catalyzed hydrolysis of ATCh. The relative free energies were determined by the QM/MM-FE calculations at the MP2/6-31+G*:AMBER level, excluding the zero-point and thermal corrections for the QM system. Values in the parenthesis are relative free energies including the zero-point and thermal corrections for the QM subsystem.

Figure 4

In substrate activate phase, an additional substrate ATCh molecule binds with the peripheral anionic site of BChE and interacts with the QM part of BChE by electrostatic interaction. Atoms of the QM part are rendered in balls and sticks. Atoms of the additional ATCh and Asp70 (D70) in the peripheral anionic site are rendered in sticks. Other atoms of BChE are rendered in cartoon of lines. See caption of Figure 1 for the color codes for different types of atoms.

Scheme 1

Proposed catalytic mechanism for BChE-catalyzed hydrolysis of acetylthiocholine