The Deacylation Step of Acetylcholinesterase: Computer Simulation Studies

Abstract

The deacylation step of acetylcholinesterase was simulated using the empirical valence bond (EVB) method in combination with free energy perturbation calculations. Before the enzyme structure was used to simulate the reaction, the protonation pattern of the acylated enzyme and the free enzyme was determined by a Monte Carlo titration. As a result, it was found that Glu199, which is located close to the catalytic triad, is protonated in the free and acylated enzyme. Also, the EVB simulation of the reaction showed that the uncharged Glu199 is favorable to stabilize the transition state of the deacylation step. This is in agreement with experiments demonstrating that the Glu199Gln mutation does not have a significant influence on the kinetics of deacylation. The EVB calculations yielded an energy barrier of the deacylation step that is 11−12 kcal/mol lower in AChE as compared to a reference reaction in water. The largest calculated rate of the deacylation reaction is kcat = 5.5 × 102 s-1 and thus only by a factor of 30 smaller than the experimental value.