Origin of the Catalytic Power of Acetylcholinesterase:  Computer Simulation Studies

Abstract

The energetics of the acylation step of AChE (acetylcholinesterase) is explored by using molecular simulation approaches. These include the evaluation of activation free energies by using the empirical valence bond (EVB) potential surface and an all-atom free energy perturbation (FEP) approach, as well as estimates of the catalytic effect of the enzyme by using the semimicroscopic version of the Protein Dipoles Langevin Dipoles (PDLD/S) method. The determination of the effect of the enzyme is based on the use of reliable experimental information in evaluating the energetics of the reference reaction in water and then on using robust simulations for the evaluation of the effect of moving the reacting system from a solvent cage to the protein active site. This procedure reduces the error range of the overall analysis since the energetics in water is not evaluated by a first principle approach. The use of two simulation methods and different initial conditions provide a way for assessing the error range of the calculations and the validity of the corresponding conclusions. Both the EVB and PDLD/S approaches show that the enzyme reduces the activation barrier of the acylation step by 10−15 kcal/mol relative to the corresponding reference reaction in water. This corresponds to a (107−1011)-fold rate acceleration, which is in good agreement with the corresponding experimental estimate. The origin of the catalytic power of the enzyme appears to be associated with electrostatic stabilization of the transition state. This electrostatic effect can be classified as a combination of reduction of the energy of the charged intermediate and reduction in the reorganization energy. The contributions of different protein residues to the stabilization of the transition state are estimated. It is demonstrated that, in contrast to some proposals, AChE and other enzymes do not work by providing a hydrophobic environment but rather a polar environment. This work concludes that the most important catalytic effects are associated with nearby residues rather than distant ionized residues. It is also concluded that the enzyme has evolved first to optimize the speed of the actual bond breaking/bond making chemical processes and only then to fine-tune the rate by optimizing the barrier for the diffusion step. Since the optimization of the chemical step involves more than 10 kcal/mol and the optimization of the diffusion step involves at most 1 or 2 kcal/mol, it appears that the possible acceleration of the diffusion step is a second-order effect. These conclusions are consistent with the available experimental studies.

Organophosphorus compounds as chemical warfare agents: a review

“…The O γ -atom of the Ser-200 residue is located 4 Å above the bottom of the gorge, 38 whose wall is aligned by 14 highly conserved aromatic residues that interact with quaternary ammonium ions, and also by a few acidic residues. 38,44,45 Moreover, the distribution of charged residues in AChE results in a permanent dipole moment that is aligned with the axis of the active site gorge; this characteristic facilitates the attraction of a positively charged substrate, guiding it through the gorge. 46 The direct interaction of the substrate with the negatively charged residues situated in the gorge is shielded by the side chains of the aromatic residues.…”

“…The stabilization of existing charges in the transition state results in the exceptional catalytic power of AChE. 43,45,51,[54][55][56][57] This mechanism is represented in Figure 4. His-440 has a high mobility during the catalytic action of AChE, 58 and its exact positioning is fundamental for achieving optimal catalytic activity.…”

“…53 The residues Trp-84, Glu-199 and Phe-330 are the main components of the anionic subsite (also known as choline binding site), strongly interacting with the quaternary ammonium group of ACh. 45,[61][62][63] Particularly, Trp-84 is very close (3.7 Å) to the ammonium group, and both Trp-84 and Phe-330 interact with this group by cation-π interactions. 64 Although used for long time, the expression "anionic subsite" is not precise, since it contains, at most, one formal negative charge; the cation-π interactions are responsible for the binding of choline to the active site, and not any eventual ionic interaction.…”

“…The NH groups of these residues' backbones hydrogen-bond to the carbonyl. 38,45,54,55,65 The residues in the oxyanion hole and in the acyl pocket take part in the stabilization of acylation and deacylation intermediates, accommodating them without the need of major conformational changes. 68 The peripheral anionic site is located near the ridge of the active site gorge and, although is not directly involved in AChE catalytic activity, comprises several superposed sites for the binding of allosteric inhibitors and activators.…”

The Contribution of Molecular Modeling to the Knowledge of Pesticides

“…The catalytic site is inside a gorge of about 20 Å; the catalytic cycle involves a catalytic triad composed of three residues Ser203, His447, and Glu334 in mouse AChE. The oxyanion hole composed of Ala204, Glu121, and Glu122 is very important as it activates the substrate via hydrogen bonds (Warshel et al 1989;Fuxreiter & Warshel, 1998). Disfunctions of AChE due to organophosphorous compounds are a major threat because they inhibit AChE irreversibly leading to convulsions, and possibly death by asphyxiation.…”