ABSTRACT The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by
rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and
narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so
deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment.
Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional
molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the
active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via
random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of
the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits
most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of
multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.