Pathways of ligand clearance in acetylcholinesterase by multiple copy sampling

The clearance of seven different ligands from the deeply buried active-site of Torpedo californica acetylcholinesterase is investigated by combining m ultiple copy sampling molecular dynamics simulations, with the analysis of protein-ligand interactions, protein motion and the electrostatic potential sampled by the Ligand copies along their journey outwards. The considered ligands are the cations ammonium, methylammonium, and tetramethylammonium, the hydrophobic methane and neopentane, and the anionic product acetate and its neutral form, acetic acid. We find that the pathways explored by the d ifferent ligands vary with ligand size and chemical properties. Very small ligands, such as ammonium and methane, exit through several routes. One inv olves the main exit through the mouth of the enzyme gorge, another is throu gh the so-called back door near Trp84, and a third uses a side door at a di rection of approximately 45 degrees to the main exit. The larger polar liga nds, methylammonium and acetic acid, leave through the main exit, but the b ulkiest, tetramethylammonium and neopentane, as well as the smaller acetate ion, remain trapped in the enzyme gorge during the time of the simulations . The pattern of protein-ligand contacts during the diffusion process is hi ghly non-random and differs for different ligands. A majority is made with aromatic side-chains, but classical H-bonds are also formed, in the case of acetate, but not acetic acid, the anionic and neutral form, respectively, of one of the reaction products, specific electrostatic interactions with p rotein groups, seem to slow ligand motion and interfere with protein flexib ility; protonation of the acetate ion is therefore suggested to facilitate clearance. The Poisson-Boltzmann formalism is used to compute the electrost atic potential of the thermally fluctuating acetylcholinesterase protein at positions actually visited by the diffusing Ligand copies. Ligands of diff erent charge and size are shown to sample somewhat different electrostatic potentials during their migration, because they explore different microscop ic routes. The potential along the clearance route of a cation such as meth ylammonium displays two clear minima at the active and peripheral anionic s ite. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both dir ections, being of the order of the ligand kinetic energy. The peripheral si te thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release. (C) 2000 Academic Pr ess.