A computational study of the deacylation mechanism of human butyrylcholinesterase.

To investigate the mechanism of the deacylation reaction in the active site of human butyrylcholinesterase (BuChE), we carried out quantum mechanical (QM) calculations on cluster models of the active site built from a crystallographic structure. The models consisted of the substrate butyrate moiety, the catalytic triad of residues (Ser198, Glu325, and His438), the "oxy-anion hole" (Gly116, Gly117, and Ala199), the side chain of Glu197, four water molecules, the side chain of Ser225, and the peptide linkage between Val321 and Asn322. Analyses of the equilibrium geometries, electronic properties, and energies of the QM models gave insights into the catalytic mechanism. In addition, the QM calculations provided the data required to build a molecular mechanics representation of the reactive BuChE region that was employed in molecular dynamics simulations followed by molecular-mechanics-Poisson-Boltzmann (MM-PB) calculations. Subsequently, we combined the QM energies with average MM-PB energies to estimate the free energy of the reactive structures in the enzyme. The rate-determining step corresponds to the formation of a tetrahedral intermediate with a free-energy barrier of approximately 14.0 kcal/mol. The modulation of the BuChE activity, exerted by either neutral molecules (glycerol, GOL) or a second butyrylcholine (CHO) molecule bound to the cation-pi site, does not involve any significant allosteric effect. Interestingly, the presence of GOL or CHO stabilizes a product complex formed between a butyric acid molecule and BuChE. These results are in consonance with the crystallographic structure of BuChE, in which the catalytic Ser198 interacts with a butyric fragment, while the cation-pi site is occupied by one GOL molecule.