Simulations of acetylcholinesterase in the presence of its inhibitors

Nearly two decades have elapsed since the first crystal structure of acetylcholinesterase-an enzyme with a nearly diffusion-controlled catalytic rate-revealed a catalytic triad at the base of a 20 A deep gorge. The crucial questions of substrates access and product release from the active site still remain active topics of research interest. The work presented here begins with an investigation of the dynamics of ligand barrier crossing inside the gorge. Molecular dynamics simulations of tetramethylammonium crossing the gorge bottleneck were conducted using umbrella potential sampling and activated flux techniques. The low energy barrier in the bottleneck region obtained from this study is consistent with the fast reaction rate of acetylcholinesterase observed experimentally. Local conformational fluctuations of the gorge residues and larger scale collective motions of the protein are found to highly correlate with the ligand crossing.; The neurotoxin fasciculin tightly binds the main gorge entrance, sterically blocking access of substrates to the active site of the acetylcholinesterase, yet kinetics experiments show some residual catalytic activity of the fasciculin bound enzyme. Analysis of the 15-nanosecond molecular dynamics trajectory of mouse acetylcholinesterase in the presence of fasciculin reveals the structural fluctuations of the enzyme are substantially increased in magnitude for the enzyme in the complex relative to a 15-nanosecond simulation of apo mouse acetylcholinesterase, particularly in the long omega loop (residues 69--96). This loop forms one wall of the active site, and the enhanced fluctuations lead to additional routes of access to the active site.; Finally, the dynamics of the encounter between fasciculin and acetylcholinesterase is influenced by conformational variations before and after binding. Sub-microsecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, were analyzed with reference to conformational changes of loop I of this three-finger toxin. The high energy barrier found between the two major substates leads to transitions that are slow on the time scale of the diffusional encounter of fasciculin and acetylcholinesterase. It seems likely that the more stable apo form binds rapidly to acetylcholinesterase; and conformational re-adjustments then occur in the resulting encounter complex.