Acetylcholinesterase, with an active site located at the bottom of a narrow and deep gorge, provides a striking example of enzymes with buried active sites. Recent molecular dynamics simulations showed that reorientation of five aromatic rings leads to rapid opening and closing of the gate to the active site. In the present study the molecular dynamics trajectory is used to quantitatively analyze the effect of the gate on the substrate binding rate constant. For a 2.4-Å probe modeling acetylcholine, the gate is open only 2.4% of the time, but the quantitative analysis reveals that the substrate binding rate is slowed by merely a factor of 2. We rationalize this result by noting that the substrate, by virtue of Brownian motion, will make repeated attempts to enter the gate each time it is near the gate. If the gate is rapidly switching between the open and closed states, one of these attempts will coincide with an open state, and then the substrate succeeds in entering the gate. However, there is a limit on the extent to which rapid gating dynamics can compensate for the small equilibrium probability of the open state. Thus the gate is effective in reducing the binding rate for a ligand 0.4 Å bulkier by three orders of magnitude. This relationship suggests a mechanism for achieving enzyme specificity without sacrificing efficiency.Rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE; EC 3.1.1.7) is essential for cholinergic neurotransmission. This importance is underlined by the large value of(1-3), which ranks as one of the highest catalytic efficiencies known (4). This magnitude of k cat ͞K m is striking for AChE, considering the fact that the substrate has to find the active site at the bottom of a narrow but 20-Å deep gorge (5) by diffusion. What is even more remarkable is the fact that, in the x-ray structure of the enzyme (5), the aromatic rings of Tyr-121 and -334 and Phe-290, -330 and -331 completely block the entrance of a 2.4-Å sphere modeling acetylcholine at Ϸ12 Å from the bottom of the gorge. These rings must reorient to temporarily make way for the incoming substrate (see Fig. 1). Molecular dynamics (MD) simulations (6) demonstrated that, by such reorientation, passage of molecules having the size of acetylcholine is allowed only a small fraction of the time (see Fig. 2). How can the enzyme retain such a high catalytic efficiency?It was recognized long ago that substrate binding could be slowed down by dynamic modulation of the active site accessibility (7), which may be called conformation gating. In one extreme, conformation gating simply turns the active site from reactive to inert toward the substrate and vice versa, but the enzyme is otherwise identical in the two states. If the switching between the reactive and inert states is stochastic (with rates w r and w i , respectively), Szabo et al. (8,9) found that the substrate binding rate constant under conformation gating is given bywhere k is the rate constant when the active site is reactive all the time and wk(w) is a relate...

We have studied the dynamic properties of acetylcholinesterase dimer from Torpedo californica liganded with tacrine (AChE-THA) in solution using molecular dynamics. The simulation reveals fluctuations in the width of the primary channel to the active site that are large enough to admit substrates. Alternative entries to the active site through the side walls of the gorge have been detected in a number of structures. This suggests that transport of solvent molecules participating in catalysis can occur across the porous wall, contributing to the efficiency of the enzyme.