Conformation gating as a mechanism for enzyme specificity

Abstract

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...

Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

“…Fast conformational changes (e.g., those on a subnanosecond time scale) are shown to have very little effect on the binding rate (22,23). That our k a calculations implicate the unbound conformation for the SRL tetraloop in the transient complex suggests that, without interactions with restrictocin, the base flip can occur, if at all, only on a slow time scale (e.g., microseconds or longer).…”

“…The effect of conformational changes on the binding rate constant critically depends on their time scales (22). Fast conformational changes (e.g., those on a subnanosecond time scale) are shown to have very little effect on the binding rate (22,23).…”

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

“…2,12,67 It should be noted internal dynamics of the protein or RNA occurring on the sub-nanosecond time scale has little impact on the binding rate. 68,69 It is interesting to note that, despite the large magnitude of the net charge on the RNA (−27e; pairing with a partner with a net charge of +6e), the magnitudes of <U el >* found here for the U1A-U1SLII system are comparable to those found for protein-protein pairs exhibiting charge complementarity. 22,23 This points to the dominant roles of specific interactions in electrostatic rate enhancement.…”

Latrunculin Analogues with Improved Biological Profiles by “Diverted Total Synthesis”: Preparation, Evaluation, and Computational Analysis

“…[40] A dynamic modulation of the binding mechanism (conformation gating) has been shown previously to affect the selectivity of some enzymes. [41,42] The role that such a mechanism plays in the binding of 1 and its analogues is the focus of current work.…”