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. 2025 Sep 19;34(10):e70297. doi: 10.1002/pro.70297

Acetylcholinesterase: Structure, dynamics, and interactions with organophosphorus compounds

Li‐Wei Hung 1,✉, Karissa Y Sanbonmatsu 2, Robert F Williams 1, Julian C‐H Chen 1,✉
PMCID: PMC12447245  PMID: 40970461

Abstract

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh), removing it from the synaptic cleft after the transmission of an electrical signal, making it an essential component of chemical neurotransmission. AChE is a serine hydrolase, containing a catalytic triad of Ser/His/Glu. AChE is a prime target for pharmaceuticals treating a variety of neurological disorders. It is also the target of synthetic organophosphorus (OP) compounds that have been used as pesticides and chemical warfare agents. OP compounds contain a potent leaving group, such as fluorine, and act by forming a covalent adduct with the catalytic serine of the AChE active site. A wealth of structural information is available for AChE, including over 300 structures, including a subset of structures in complex with drugs as well as OP compounds. This review will highlight the interactions between OP compounds and AChE from a structural and computational perspective, with a discussion of access to the active site, as well as side reactions that lead to dealkylation of the OP‐catalytic serine adduct, a process known as aging. We conclude that while the majority of the conformational changes needed to accommodate the OP compounds are localized to the acyl loop in the crystal structures, molecular dynamics simulations highlight the potential for a far more dynamic enzyme.

Keywords: acetylcholinesterase, molecular dynamics, nerve agent, organophosphorus, protein dynamics, structure

1. BACKGROUND

Acetylcholinesterase (AChE) is a serine hydrolase critical for neural signaling. AChE hydrolyzes the neurotransmitter acetylcholine (ACh) into choline and acetic acid, a process essential for terminating synaptic transmission at cholinergic synapses and thus regulating the duration and magnitude of cholinergic neurotransmission in both the central and peripheral nervous systems (Figure 1). AChE has a very high catalytic activity—each molecule of AChE hydrolyzes about 5000 ACh per second (Potter et al., 1984), approaching the limit allowed by diffusion of the substrate (Quinn, 1987; Taylor & Radic, 1994). Through this enzymatic action, AChE ensures the precise spatiotemporal control of cholinergic signaling, facilitating the transition from excitation to quiescence and enabling the fine‐tuning of neuronal circuits underlying cognition, movement, and autonomic functions.

FIGURE 1.

FIGURE 1

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(a) Reaction catalyzed by AChE. The neurotransmitter acetylcholine is split into acetic acid and choline, with the addition of H2O. (b) Hydrogen bonding (dotted lines) and hydrophobic interactions between Y337, F338, and F295 (dashed lines) within the active site. Key residues are marked with hAChE (4EY4) residue numbering. (c) Catalytic cycle of AChE showing proton transfer between key residues and the substrate.

Because of the pivotal role that AChE plays in the nervous system, the enzyme has long been an attractive target for the rational design of mechanism‐based inhibitors (Quinn, 1987). Many chemicals affect the activity of AChE through reversible or irreversible inhibition of AChE's function, increasing the level and duration of ACh action. Several FDA‐approved drugs target AChE in a reversible manner, as a therapeutic for Alzheimer's disease (Alhazmi & Albratty, 2022), including donepezil (Aricept), rivastigmine (Exelon), galantamine (Razadyne), and as of July 2024, Zunveyl, which is a prodrug of galantamine (Baakman et al., 2016; Li et al., 2025). The interactions of AChE with several reversible inhibitors have been summarized in a recent review (Luque & Muñoz‐Torrero, 2024). In contrast, toxic effects are associated with irreversible modulators of AChE activity. Notably, these include organophosphorus (OP) compounds, which exert their toxic effects by irreversibly inhibiting AChE through covalent modification of the enzyme. The inhibition of AChE activity over time causes a rapid buildup of excess ACh in the synaptic cleft, leading to continuous depolarization of the postsynaptic neurons with concomitant acute cholinergic and muscarinic toxicity (Aroniadou‐Anderjaska et al., 2023; Kovarik et al., 2024; Vale et al., 2016; Voros et al., 2024).

Before the first complete amino acid sequence of an AChE (Schumacher et al., 1986) was determined in the mid‐1980s, the unique biochemical properties and enzymatic kinetics had already been extensively probed (Quinn, 1987; Taylor et al., 1986). Following the first crystal structure of AChE from Torpedo californica determined in 1991 (Sussman et al., 1991), AChE structures from Homo sapiens (Kryger et al., 2000), Mus musculus (Bourne et al., 1995), Electrophorus electricus (Bourne, Grassi, et al., 1999), venom of Bungarus fasciatus (Bourne et al., 2015), and Drosophila melanogaster (Nachon et al., 2020) have been determined and extensively studied. The protein sequences are highly conserved among vertebrate AChEs but marginally conserved with the Drosophila enzyme (Figure 2 and Table 1), consistent with their evolutionary distances. Despite the borderline sequence identity between Drosophila and the other AChEs, their core structures can be superposed very well (Wiesner et al., 2007) (Figure 3 and Table 2).

FIGURE 2.

FIGURE 2

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Multiple sequence alignment of AChE species with structures reported in the PDB. The three residues highlighted in red boxes are the catalytic residues S, E, and H.

TABLE 1.

Pairwise sequence identity (%) of AChE with crystal structures (HUMAN: Homo sapiens; MOUSE: Mus musculus; ELEEL: Electrophorus electricus; BUNFA: Bungarus fasciatus venom; TORCA: Torpedo californica; DROSO: Drosophila melanogaster).

HUMAN MOUSE ELEEL BUNFA TORCA DROSO
HUMAN 87.45 87.17 65.09 58.65 38.01
MOUSE 98.51 63.86 59.29 39.74
ELEEL 63.64 58.92 39.74
BUNFA 66.17 41.25
TORCA 38.50
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FIGURE 3.

FIGURE 3

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Structural superposition of AChEs from different organisms.

TABLE 2.

Pairwise Cα RMSD (Å) of AChE structures from different organisms.

HUMAN MOUSE ELEEL BUNFA TORCA DROSO
HUMAN 0.69 0.806 0.975 1.08 1.75
MOUSE 0.486 1.01 0.913 1.62
ELEEL 1.07 0.968 1.81
BUNFA 0.902 1.95
TORCA 1.69
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This review focuses on the structural aspects of AChE and the interaction of OP compounds with AChE, including the aging reaction, which is a formal dealkylation reaction of the OP‐inhibited AChE. ‘Aged’ AChE cannot be reactivated directly by traditional treatments of oximes or related nucleophilic reactivators, and replacement AChE must be re‐synthesized over time to restore full cellular function. This review will also evaluate the dynamic aspects of AChE, which have been found to be important for native substrate accessibility as well as the accessibility of OP compounds and nucleophilic oxime countermeasures into the active site, and the critically important allosteric interactions required for AChE function.

2. STRUCTURE OF AChE

Crystal structures of AChEs from six different organisms have been determined: human, mouse, electric eel, electric ray, fruit fly, and one from snake venom. These crystal structures reveal a protein consisting of a 12‐stranded central mixed β‐sheet surrounded by 14 α‐helices forming an extended α/β hydrolase fold (Ollis et al., 1992). The most significant feature of this structure is a narrow and deep gorge, called the active‐site gorge (Dvir et al., 2010), which contains the catalytic triad near its base. The gorge is nearly 20 Å long and 5 Å wide with a wider opening tunneling beyond the center of this slightly ellipsoid‐shaped (~ 45 Å × 60 Å × 65 Å) enzyme (Figure 4). The active site of AChE is located at the lower section of the active‐site gorge and is lined by multiple, highly conserved aromatic residues. The active‐site gorge can be subdivided into several sub‐sites: (1) The catalytic triad consists of residues Ser203, His447, and Glu334 (Rachinsky et al., 1990) (Note: the residue numbering hereafter will correspond to the human AChE (hAChE) numbering system. Respective residue numbers for the other AChE sequences are listed in Table 3) and is not easy to access because it is buried at the base of the gorge with the catalytic serine ~4 Å above the base of the gorge. The narrowest section of the active site gorge is close to the size of the natural substrate acetylcholine; (2) the catalytic aromatic site (Sussman & Silman, 2020) (CAS, also known as catalytic anionic site, or anionic site) is composed of conserved aromatic residues (Trp86, Tyr133, Tyr337, and Phe338) and mediates the binding of the positively charged choline moiety of the substrate via a cation‐π interaction with Trp86 (Luque & Muñoz‐Torrero, 2024); (3) the acyl‐binding pocket (Phe295, Phe297, and Trp236) is responsible for substrate selectivity by preventing access to the larger members of the choline ester series and by stabilizing the acetyl group of ACh; and (4) the oxyanion hole (Gly121, Gly122, and Ala204) which provides hydrogen bond donors to stabilize the transition state of the substrate. These four sites are critical for enzyme catalysis and their functions are easily rationalized based on the structural information gained from X‐ray crystallography (Figure 4).

FIGURE 4.

FIGURE 4

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Top left: Ribbon trace of hAChE, with the N‐ and C‐terminal identified. Top right: Surface view with the active site residues of the catalytic site (CS; Ser203, His447, Glu334) in magenta. Bottom left: View of the active site gorge with the peripheral aromatic site (PAS) in green, acyl binding site in yellow, anionic site in cyan, and oxyanion hole in blue and the representative residues are labeled. Bottom right: View of active site gorge with the “side door” labeled in lime green and the “back door” labeled in orange.

TABLE 3.

AChE functional sites and corresponding residues.

CS CAS Acyl binding pocket Oxyanion hole PAS
Human S203, H447, E334 W86, Y133, Y337, F338 F295, F297, W236, F338 G121, G122, A204 Y72, D74, Y124, W286, Y341
Mouse S203, H447, E334 W86, Y133, Y337, F338 F295, F297, W236, F338 G121, G122, A204 Y72, D74, Y124, W286, Y341
Electric Eel S203, H447, E334 W86, Y133, Y337, F338 F295, F297, W236, F338 G121, G122, A204 Y72, D74, Y124, W286, Y341
Torpedo S200, H440, E327 W84, Y130, F330, F331 F288, F290, W233, F331 G118, G119, A201 Y70, D72, Y121, W279, Y334
Drosophila S238, H480, E367 W83, Y162, Y370, F371 L328, F330, W271, F371 G150, G151, A239 E69, Y71, M153, W321, Y374
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Removed from the active site region is the peripheral aromatic site (PAS, also referred as peripheral anionic site [Sussman & Silman, 2020]), which is an additional binding site for ACh and for other quaternary ligands (Bergmann et al., 1950; Changeux, 1966; Mooser & Sigman, 1974). In mammalian AChE, the PAS, consisting of residues Tyr72, Asp74, Tyr124, Trp286, and Tyr341, is located at the entry to the gorge. It has been demonstrated that ACh is transiently bound to the PAS in the first step of the catalytic pathway (Szegletes et al., 1999). It has also been suggested that the PAS has a significant role in allosteric regulation of AChE‐catalyzed hydrolysis (Bourne et al., 2003); however, the molecular mechanism that couples this site to the active center in the gorge base and modulates the catalysis remains unclear. The PAS has also been proposed in heterologous protein associations with AChE (Bourne, Taylor, et al., 1999). In contrast, although Drosophila AChE (DmAChE) shares marginal sequence identities with hAChE and mAChE, the residues in the AChE functional sites are all very well conserved, with the exception of Glu69, Tyr71, and Met153 of the Drosophila PAS. These differences influence both the steric and electrostatic properties of the entrance to the active site gorge and suggest that the catalytic properties of AChE have been conserved throughout the evolution of this enzyme. Consequently, the PAS may play an important role in the substrate specificity of AChEs across species (Wiesner et al., 2007).

A considerable portion of the active‐site gorge is formed by residues from the long omega (Ω) loop (Cys69‐Cys96 in hAChE). Conformational mobility of the surface of the Ω‐loop was implicated by substrate accessibility to the active center and by the mechanism of allosteric modulation of enzymatic activity. Experimental studies (Shi et al., 2001; Shi et al., 2002) have shown that the conformational variations of this loop are associated with ligand binding, and molecular dynamics (MD) simulations suggest that enhanced flexibility of the long Ω‐loop leads to additional routes of access to the active site (Bui et al., 2004).

As an enzyme that exists in eukaryotic organisms, it is not surprising that there are a series of N‐linked glycosylation sites that likely contribute to the stability of the enzyme; however, these sites are not directly involved in enzymatic activity. Furthermore, the C‐terminal region of AChE anchors the enzyme to the membrane, either by GPI‐mediated attachment or through a protein–protein interaction with accessory proteins such as the protein proline‐rich membrane anchor (PRIMA) (Chen et al., 2011).

3. AChE‐OP INTERACTIONS

OP compounds that are toxic and bind to AChE contain phosphorus with diverse structures (P(=O, =S)–X, –Y, −Z where X = O, C, S, N; Y=O, C, S, N; and Z = F, Cl, CN, S, O) that are generally derived from phosphates and phosphonates as shown in Figure 5. OPs are highly effective pesticides and insecticides, and many have been developed and bioengineered to be highly toxic, very effective inhibitors of hAChE. These compounds have been weaponized and used in military operations as well as assassinations. Since these toxic compounds interfere with the function of AChE, they have been commonly referred to as nerve agents (Young & Watson, 2020). Some are well‐known nerve agents, such as sarin or VX (Delfino et al., 2009) (see Figure 5), but others, such as the Novichoks or A‐agents (Kloske & Witkiewicz, 2019; Morato et al., 2025; Nepovimova & Kuca, 2020), while more toxic, are less well known. Despite the ratification of the Chemical Weapons Convention over two decades ago, the potential for misuse of these compounds and their derivatives highlights the ongoing threat to military personnel and civilians and makes our understanding of how they interact with hAChE critically important to counter their threat and develop next‐generation countermeasures and antidotes. This section presents an overview of the interactions between AChE and OP compounds. Specific interactions of the OP moieties with the active site residues of AChE and their reaction mechanisms are discussed in detail elsewhere (Gupta, 2020; Kloske & Witkiewicz, 2019; Lushchekina & Masson, 2018; Quinn et al., 2017; Silman & Sussman, 2017; Zlobin et al., 2024).

FIGURE 5.

FIGURE 5

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Chemical structures of selected OP compounds discussed in this article.

The chemical structures of OP compounds (vide supra and Figure 5) play a critical role in dictating their reactivity towards hAChE and determine their overall toxicity profile (Sirin et al., 2012). Structural features, such as the leaving group (Z), alkyl chain length (X), and substituent groups (Y), influence the rate of OP binding to hAChE, as well as the stability and pharmacokinetic properties of the resulting enzyme‐inhibitor complexes. The kinetics of the OP‐hAChE interaction involve multiple steps, including reversible binding, phosphylation of the catalytic serine on hAChE, and aging (for example, when the loss of an alkyl side chain attached to the phosphorus through an oxygen leaves the oxygen anion attached to the phosphorus), each influencing the potency and duration of enzyme inhibition. Initially, OPs transiently associate with hAChE through non‐covalent interactions, allowing for the formation of reversible enzyme‐inhibitor complexes. Subsequent nucleophilic attack by the serine residue at the active binding site leads to the formation of a covalent bond between the phosphorus of the OP and the oxygen of the serine at the hAChE active site with concomitant loss of a leaving group (Z) such as the fluorine in the case of sarin, or the alkyl sulfur moiety in the case of VX. The phosphylated enzyme is no longer capable of hydrolyzing ACh, and dephosphylation by water can be very slow, on the order of days, depending on the structure of the OP. Medical treatments for inhibition of hAChE by OP compounds typically employ strongly nucleophilic oximes, such as 2‐pyridine aldoxime methyl chloride (2‐PAM), that attack the phosphorus bound to the hAChE, regenerating the active enzyme.

If hAChE is not reactivated by an oxime nucleophile, the phosphylated hAChE can undergo a further dealkylation reaction, known as aging (Figure 6). This results in a complex that is refractory to reactivation by oximes (Millard et al., 1999; Sirin et al., 2012). The difficulty in reactivation is not only because the complex is more stable, forming a salt bridge with His447, but also because there are no available nucleophiles (e.g. water, OH−, or even oxime) strong enough to attack the phosphorus and displace the serine, regenerating hAChE. The dealkylation reaction leaves an oxygen anion on the phosphorus, increasing the electron density of the phosphorus and making it resistant to an attack by any nucleophile. The rate of aging is proportional to the electron‐donating capacity of the alkyl group (Sun et al., 1979), which varies among different OPs (Worek & Thiermann, 2013) (Table 4), influencing the efficacy of potential antidotes and treatment strategies targeting OP poisoning. An approach to realkylate the aged adduct followed by reactivation with oxime nucleophiles has been proposed (Topczewski & Quinn, 2013). However, the aged OP adduct was remarkably unreactive, thus requiring further research to solve this conundrum (Quinn et al., 2017).

FIGURE 6.

FIGURE 6

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Examples of aging reactions of AChE and representative nerve agents (GA, GB, VX).

TABLE 4.

Aging half‐time of human AChE inhibited by selected OP pesticides and nerve agents.

OP compound Half‐time (hours)
Tabun (GA) 19.2
Sarin (GB) 3.0
Soman (GD) 0.07
Cyclosarin (GF) 7.0
VX 36.5
VR 138.6
CVX 32.2
Paraoxon‐ethyl 31.5
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Note: Data taken from Worek et al. (1999, 2004, 2008).

4. STRUCTURAL ASPECTS OF AChE AND OP INTERACTIONS

Structures of Torpedo, mouse, and human AChEs (TcAChE, mAChE, and hAChE, respectively) after equilibration with several types of nerve agents and OP‐based pesticides have been determined in aged and non‐aged states (Table S1). In addition, a number of structures have been solved in complex with known oxime molecules, such as HI‐6 and 2‐PAM (Allgardsson et al., 2016; Bester et al., 2018; Bester et al., 2019; Ekström et al., 2007; Ekström et al., 2009; Franklin et al., 2016; Gerlits et al., 2021; Gorecki et al., 2020; Katz et al., 2015; Lindgren et al., 2022; McGuire et al., 2021; Sanson et al., 2009; Santoni et al., 2018), providing the structural foundation of effective treatments for OP poisoning.

When an OP interacts with the active site of an AChE, the phosphorus atom forms a covalent bond to the serine oxygen (Ser203 in hAChE and mAChE, subsequent residue numbering will refer to the corresponding residues in hAChE), with concomitant loss of a reactive leaving group such as a fluorine for sarin or soman, leading to the inhibition of hAChE activity. The covalently bound OP causes conformational changes in the active‐site gorge primarily dependent upon the size of the OP compounds' side chains. Most OP compounds covalently bind to the catalytic site with their larger substituents oriented towards the choline‐binding site, and the smaller substituent facing the acyl‐binding pocket, similar to that of ACh (Sussman et al., 1991). The overall structures of these OP/hAChE complexes are very similar to the corresponding unliganded structures, with specific conformational changes discussed below.

Most OP compounds (e.g., sarin, tabun, VX, A‐230, etc.) contain a chiral phosphorus stereocenter and exist as racemic mixtures. The Sp(−) optical isomer is the more active enantiomer and has a faster rate of reaction and greater binding affinity to AChE than the Rp(+) enantiomer. However, the OP compounds with additional chiral centers in their side chains can have varying activity and toxicity profiles (Benschop & De Jong, 1988). One example is soman, which exists as four diastereomers due to the chiral carbon on the pinacolyl side chain. Crystal structures are assumed to report the isomer preferentially bound to AChE (Bester et al., 2018).

4.1. Active site

The overall configuration of the Ser203‐OP structure is very similar for all AChE‐OP structures, with the phosphyl oxygen hydrogen bonding to the main chain amide nitrogen of the oxyanion hole residues, Gly121, Gly122, and Ala204. The tight coordination of the oxygen places one of the phosphyl substituents in the acyl pocket comprised of amino acids Phe295, Phe297, Trp236, and Phe338, while the other substituent is directed towards the indole ring of Trp86. The oxygen of the substituent (e.g., the ethoxy group in tabun or the isopropoxy moiety in sarin) is involved in hydrogen bonding with His447. This interaction breaks the hydrogen bonding network in the catalytic triad and causes the His447 side‐chain imidazole ring to reorient depending on the size of the phosphyl substituents. This conformational change is coupled with a similar side‐chain movement of Phe338 due to steric strain. In general, structures of OP adducts with smaller substituents, such as aged sarin, methaminophos (MeP), and VX, lead to a very small side‐chain shift of His447 compared to their non‐aged forms (Hörnberg et al., 2007). As such, the catalytic center largely resembles that of the unliganded forms. In contrast, the phosphyl substituents of tabun, DFP, and non‐aged VX cause His447 to undergo a side‐chain flip of 180° and a rotation from 65° to 105° around its chi‐1 axis for the corresponding hydrogen bond to form. The adduct of fenamiphos (FeP) and mAChE shows a different arrangement of its phosphyl substituents where its large amide moiety points towards Trp86 and His447 but does not directly interact with His447. Structural alignment of the AChE molecules in crystal structures of adducts of mAChE with RVX (3ZLT) and GF (3ZLU) shows that the side chains of His447 and Phe338 adopt a range of conformations, suggesting conformational mobility is a common feature in nerve agent adducts of AChE (Artursson et al., 2013).

4.2. Substrate and solvent access to the active site

The catalytic site is located deep within the enzyme, raising questions about substrate and solvent accessibility to the active site. To better understand the routes of access by substrates, and the subsequent exit routes from the active site after hydrolysis, existing crystal structures of hAChE were subject to additional refinement using PDB_REDO (Joosten et al., 2014). The two monomers present within the asymmetric unit, although similar in structure, show minor conformational changes dependent upon the bound ligand, painting a more dynamic picture of the gorge. A notable example is found in complexes of paraoxon with hAChE, where in the PDB 5HF8 monomer B structure, the loop consisting of residues 289–297 is displaced, leading to a second pathway to the active site through an apparent concerted motion of Phe297 and Glu285 side chains.

The gorge is lined with solvent molecules, with a trend towards more fixed positions in the deeper parts closer to the catalytic site. An overlay of representative OP agents bound to human AChE is shown in Figure 7. As discussed earlier, the larger substituents occupy the choline binding site, such as the A‐type agents and the cyclohexyl moiety of cyclosarin. These side chains displace solvent molecules in the vicinity. However, within the gorge, there are sites where the solvent molecules are in nearly identical positions regardless of the ligand that is bound, including a water that acts as a hydrogen bond donor to the main chain carbonyl of Thr83.

FIGURE 7.

FIGURE 7

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Representative structures of A‐type, G‐type, and V‐type OP nerve agents bound to human AChE. (a) A‐230/hAChE (6NTO, monomer B); (b) Tabun (GA)/hAChE (6WUV, monomer A); (c) Cyclosarin (GF)/hAChE (6WVP, monomer B); and (d) VX/hAChE (6CQZ, monomer B).

4.3. Acyl binding pocket

The acyl binding pocket is much smaller in volume compared to the choline binding site because of the size and shape of AChE's natural substrate, ACh. Conformational changes of the acyl binding pocket loop occur to accommodate OP compounds, as a consequence of larger phosphyl moieties. Analysis of crystal structures of AChE and OP compounds reveals that the acyl loop is the most conformationally diverse subdomain around AChE's catalytic site (Radić, 2021; Radić, 2024). This strongly suggests that the conformational change in the acyl pocket plays a role in modulating the aging reaction of AChE (Hörnberg et al., 2007). Large distortions of the acyl loop induced by AChE‐OP interactions appeared to trigger homodimer dissociation in hAChE (Blumenthal et al., 2021; Radić, 2024). OP compounds with smaller substituents such as VX, soman, and tabun can be stabilized in the acyl pocket without distortion of the acyl loop (Luedtke et al., 2021). However, diisopropyl fluorophosphate (DFP), which resembles sarin, but is larger with an additional isopropyl group, is an OP insecticide and a G‐nerve agent simulant. Both non‐aged and aged DFP‐mAChE complexes are positioned with an isopropyl moiety facing the acyl loop pocket which introduces significant and similar distortions in the acyl pocket (Hörnberg et al., 2007). Acyl loop residue Pro290 makes a 5.5 Å backbone shift, along with Phe295 and Phe297, in order to accommodate the isopropyl group. Similar observations were also reported in the structure of TcAChE complexed with DFP (Szegletes et al., 1999). Significant acyl loop perturbations are observed in binary complexes of hAChE with paraoxon and appear to stem from interactions between the Phe295 phenyl ring and the ethoxy group of the adduct positioned in the acyl pocket, resulting in large displacements of Arg296 (Franklin et al., 2016) or Pro290 and Ser293 (Lushchekina & Masson, 2018) in the acyl loop. These conformational changes cause a narrowing of the gorge near the peripheral site that may impede entry of reactivator molecules inhibiting their action. However, the perturbations in the structure of the acyl loop can be partially restored upon oxime binding (Franklin et al., 2016; Gerlits et al., 2022). These large acyl loop structural perturbations appear to be specific to OP insecticides‐AChE conjugates and are not observed in AChE inhibited by traditional OP nerve agents (Gerlits et al., 2022).

4.4. Peripheral aromatic site (PAS)

The PAS is located near the opening of the gorge and plays a prominent role in substrate and ligand binding (Bourne, Taylor, et al., 1999). Five aromatic residues (Table 3) decorate the PAS, forming a low‐affinity, transitional binding site for the substrate and facilitating its transfer to the deep catalytic site. Conformational flexibility is another feature of the PAS. For example, the crystal structure of a paraoxon‐hAChE complex (POX‐hAChE), 8DT2 (Gerlits et al., 2022), demonstrates that the PAS Trp286 flips its indole ring to accommodate a larger conformational change of the acyl loop (Figure 8). This was not observed in other POX‐hAChE structures such as 5HF5 (Franklin et al., 2016), a structure which presumably consists of the same type of molecules.

FIGURE 8.

FIGURE 8

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Paraoxon/hAChE complexes, showing conformational flexibility of the acyl binding pocket. Side chain flip of F297 leads to opening of acyl binding pocket to exterior. Top: “closed” state, from 8DT2, monomer A. Bottom: “open” state, from 5HF8, monomer B. For generation of these figures, solvent molecules have been omitted.

Oxime activators such as HI‐6 have been observed to anchor on the PAS with one of their aromatic groups stacking with Trp286 and Tyr72 and creating a wide gap that allows the activator access to the active site in the hAChE GA‐HI‐6 complex structure (McGuire et al., 2021). Similar interactions are also seen in the POX‐AChE MMB4/RS‐170B structures (Gerlits et al., 2022), with the binding of the oxime partially restoring the acyl loop conformation that is found in the apo state. AChE interacts through the PAS with an amyloid β‐peptide, promoting a conformational change that accelerates Aβ aggregation and increases neurotoxicity (de Ferrari et al., 2001). AChE can also have a similar chaperoning effect on prion protein (PrP) aggregation, which may have relevance in both prions and Alzheimer's disease (Luque & Muñoz‐Torrero, 2024).

4.5. Ω‐loop

The Ω‐loop is a significant structural element in AChE, consisting of residues between two cysteines (Cys67‐Cys94 of TcAChE and Cys69‐Cys96 of mAChE or hAChE). This element plays a crucial role in maintaining the active site's architecture and accessibility. The conformations of the Ω‐loops are very consistent in all AChE crystal structures, including those complexed with ligands and OP conjugates (Radić, 2021; Radić, 2024). Nevertheless, molecular dynamics (MD) studies have indicated that the Ω‐loops are actually quite mobile (Xu et al., 2008). Experimental and theoretical studies revealed that this loop is flexible and contributes to transient gorge enlargements that may facilitate ligand binding and release (Cheng et al., 2017). Fluorescence spectroscopy with site‐directed labeling and cysteine substitution mutagenesis showed that the conformational change of the omega loop is largest near the tip of the loop (residue Glu84 on mAChE) and smallest near the hinge (residue Leu76) with the extent of the conformational change correlating with the size of the ligand. It seems that ligand binding at the catalytic site allosterically alters the conformation of a specific segment of the Ω‐loop (Küçükkilinç et al., 2010; Shi et al., 2001).

4.6. Oxime reactivation

Oximes possess a nucleophilic oxime group (‐C=NOH) that can attack the phosphorus atom of the phosphylated serine in the inhibited AChE. This highly nucleophilic moiety is capable of displacing the phosphylated serine hydroxy group and regenerating the enzyme. Prior to aging and the spontaneous elimination of the isopropoxy (e.g., sarin and DFP) or the ethoxy group (e.g., VX and tabun) in OP‐inhibited AChEs, it is possible to reactivate the enzyme with oximes. Although traditional oxime therapies were less effective in cases caused by A‐series OPs, it has been shown recently that bipyridinium‐based oximes such as HI‐6, MMB‐4, and 2‐PAM can reactivate A‐series‐adducted hAChE in vitro (Morato et al., 2025). Crystal structures of these oximes complexed with OP‐complexed AChEs provide hints on the reactivation requirements (Artursson et al., 2013; Driant et al., 2017). The oximes anchor one end to the PAS aromatic residues while extending the opposite end of the oxime towards the active site, placing it in a more favorable, productive conformation facilitating attack on the phosphorus of the complexed OP. These oxime‐assisted reactivation processes depend on the structures of oxime, and on the structure and characteristics of the OP compound and of the OP‐AChE conjugate (Kovarik et al., 2024; Kovarik & Maček Hrvat, 2020; Zorbaz et al., 2020). Using HI‐6 as an example, the overall spatial arrangement of each end of HI‐6 and the reactivation efficiency appears to rely on their ability to approach and interact with the phospho‐adduct, which, in turn, is influenced by the specific conformation and electrostatic environment of the active site. Generally, the closer HI‐6 is to the active site and the inhibitor, the more likely it will reactivate AChE due to the oxime arm interactions with the complex being in closer proximity (Bagrowska et al., 2024; McGuire et al., 2021).

However, structure–based design of oxime countermeasures has seen limited success (Kovalevsky et al., 2016). This can, in part, be attributed to the difference between static crystal structures and the dynamic nature of AChE. Over a hundred crystal structures of AChE in complex with ligands revealed surprisingly similar backbone conformations, despite considerable structural diversity of bound ligands. A fuller picture of dynamics is presented using alternative experimental methods such as fluorescence spectroscopy and MD simulation, which reveal the dynamic nature of AChE, especially in the PAS and the omega loops, which are static in crystal structures. While it is likely that efforts to design more effective oximes to reactive OP‐inhibited AChEs might have greater success when both static structures and the dynamic properties of AChE are taken into account, the aging problem with the concomitant increase in electron density at the phosphorus makes improved oximes problematic. That said, approaches to systematically optimize or to develop new classes of uncharged oxime reactivators have shown potential towards effective treatment of OP poisoning (Mlakić et al., 2021; Radić et al., 2012).

5. AChE DYNAMICS REVEALED BY MOLECULAR SIMULATIONS

AChE is one of the fastest enzymes known, hydrolyzing its substrates with a turnover rate near the limit of diffusion. The active site of AChE is embedded near the bottom of a 20 Å long and 5 Å narrow gorge. Despite the strong electrostatic environment found in the gorge, the steric environment appears to be inconsistent with the enzyme's high efficiency. In the absence of experimental evidence, computational simulations with molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) can provide clues that explain the enzyme's activity and dynamic nature that are not revealed in the snapshots obtained from crystal structures (Célerse et al., 2024; de Boer et al., 2021; Driant et al., 2017; Sussman & Silman, 2020; Xu et al., 2017). Dynamic features that can be important for the access of the substrate to the active site, as well as the exit of hydrolysis products, such as the back door or side door (Gilson et al., 1994), and the breathing motion of the gorge (Cheng et al., 2017) observed in computational simulations can help interpret data and help explain, in part, the discrepancy between structural evidence and the biochemical observations.

5.1. The back and side doors

Previous microsecond explicit solvent MD simulations of TcAChE suggested the possibility of a back door formed transiently by the rotation of Trp84 (Trp86 in hAChE) (Cheng et al., 2017). The simulations showed the opening formed is large enough to allow water molecules and small ions to pass through, suggesting an alternative pathway near AChE's catalytic site to traffic its product and solvent. The existence of this putative back door was first observed in a crystal structure of TcAChE in complex with the inhibitor aflatoxin where the side chain of Tyr442 (Tyr449 of hAChE and mAChE) located at the bottom of the active site gorge rotates to form a 3.4 Å wide channel between the catalytic center and the external surface of AChE (Sanson et al., 2011). This back door can be enlarged further with the coordinated movement of Trp84 in the Ω‐loop. The Tyr442 back door also appears in MD simulations of TcAChE identifying a possible route for the rapid clearance of the hydrolysis products from the active site that is consistent with the rapid, diffusion‐controlled enzyme turnover. In the absence of the conserved tyrosine residue, structures of DmAChE complexed with tacrine‐derived inhibitors, ZA (6XYY) or ZAI (6XYU), also showed similar open channels connecting the base of the active‐site gorge to bulk solvent (Nachon et al., 2008; Nachon et al., 2020; Sussman & Silman, 2020). The crystal structure of the complex of banded krait, Bungarus fasciatus, AChE (BfAChE) with an inhibitory monoclonal antibody fragment further supports the co‐existence of the movement between open and closed back door conformations facilitating product removal (Bourne et al., 2015).

In early explicit solvent simulations of AChE, a side door mechanism facilitating the conformational opening of a second channel was observed in studies of TcAChE and Tacrine (Wlodek et al., 1997), as well as mAChE complexed with the neurotoxin fasciculin‐2 (Fas2) (Bui et al., 2004; Tai et al., 2002). This side door is formed by side chain rotations of residues on the Ω‐loop located between Asn87 and Asp74, located near the opening of the gorge. The study, using multiple MD simulations, explored the trafficking of thiocholine (TCh), a mimic of the native product, within the active site gorge of TcAChE, suggesting multiplex exit mechanisms in AChE's catalytic cycle (Xu et al., 2010). Using a starting model from a crystal structure in which TCh (PDB 2C5G) is bound at either or both the CAS and the PAS sites, the study showed that the majority of the TCh, initially at the CAS site, exited from the catalytic site via the “back‐door” (>65%). Some TCh was retained at the CAS site (>20%), while a small amount was released through a “side‐door” of the main gorge (Xu et al., 2010). Another study, focused on the reversible binding of 2‐PAM to hAChE, used an enhanced‐sampling MD method that captures rare events, called ligand‐binding parallel cascade selection molecular dynamics (LB‐PaCS‐MD). This method involves repeating multiple short time scale MD simulations in parallel, beginning from configurations capable of inducing ligand binding or unbinding processes. This technique ranks and selects configurations as the initial structures for the next cycle from each short time scale MD simulation. This is based on the shortest distance between the center‐of‐mass of a ligand and the targeted atom or residue in a protein. The study showed that a so‐called acyl door (Tyr341) was the predominant route for binding (>60%) and unbinding (>90%) of 2‐PAM that concomitantly occurred with conformational changes of the key residues Trp86, Tyr341, and Tyr449 in the Ω‐loop (Kongkaew et al., 2024). The overall observations, interpretations, and conclusions from these MD studies support the general concept that both the substrates as well as the inhibitors enter the catalytic site via the main gorge, while small ligands or reaction products such as ammonia or methane exit the gorge via multiple pathways (Cheng et al., 2017; van Belle et al., 2000). Figure 9 shows the residues involved in various states of the back door (Trp86, Tyr449), side door (Ω‐loop in purple), and acyl door (Tyr341) pathways and their unique spatial relationships to the active site gorge.

FIGURE 9.

FIGURE 9

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Stereo illustration showing the residues involved in the states of the backdoor (W86, Y449), side‐door (Ω‐loop in purple), acyl door (Y341), and their spatial relationships to the active site gorge.

These studies raise several implications for the formation of the OP serine adduct and the subsequent aging reaction. The leaving groups associated with the initial reaction of an OP compound with the catalytic serine may exit the active site via different pathways depending on their bulkiness. For example, fluorine versus the much larger aminothiolate leaving group of VX shows a size differential requiring different exit paths from the active site for kinetic viability. Furthermore, the dealkylation reaction in aging necessitates the removal of the alkyl group, which may also leave via the back door or side door to maintain the diffusion‐controlled reaction. Future MD studies, combined with full‐length tetrameric structures, could aid in resolving these questions.

5.2. The ‘breathing’ motion

The dynamics of the coordinated motion that modulates the path of AChE's catalytic gorge were revealed in microsecond MD simulations of TcAChE in both its apo form and in complex with the drug E2020 (Cheng et al., 2017). These computational studies were among the longest MD simulations for a protein at the time of their publication, and they highlighted the potential role of allostery in the catalytic cycle of AChE. This agrees with the observations that the native biologically active form of hAChE is not monomeric. Both dimers (TcAChE) and tetramers (mAChE and hAChE) have been demonstrated (Cheng et al., 2017). However, the physiological tetrameric form is membrane‐bound and found in high local concentrations anchored in the vicinity of the post‐synaptic membrane. One of the most notable observations from these studies is the radius of the ‘bottleneck’ or the narrowest section of the gorge tunnel, defined by the spacing between Tyr121 and Phe330. In TcAChE, this spacing varied between 0.9 Å and 2.5 Å in two structural snapshots of simulations that spanned 50 ns. Based on the analysis of MD trajectories, the Ω‐loop, residues 324–400, and residues 428–450 of TcAChE are directly involved in the correlated movement. Residues linking these subdomains allosterically modulate the gorge motions via a dynamic communication pathway. These results suggest that AChE's ability to achieve such high, diffusion‐controlled kinetic efficiency, albeit with a buried catalytic center, is attributed in part to its dynamics.

The extent and frequency of the gorge breathing motion can be measured by the distribution of the radius of the bottleneck throughout the course of a simulation. These distributions generally resemble that of a Poisson distribution, suggesting the stochastic nature of these movements. However, the mean opening size of the bottleneck in a monomer, a complex, or a dimer form is all different. The apo monomer has a mean opening size of 1.67 Å, while it is 2.3 to 2.4 Å for both the apo dimer and the monomer/E2020 complexes but ~2.7 Å for the dimer/E2020 complex (Millard et al., 1999). In addition, the distributions are notably sharper for dimers in their apo form and in complexes compared to monomers. Based on MD studies, a bound inhibitor or dimerization of TcAChE not only increased the gorge radius for both monomers in the dimer and their opening rate, but also changed the dynamics of the involved subdomains.

To date, all AChE crystal structures are of a truncated construct of the enzyme in order to facilitate crystallization and high‐resolution structure determination. As a result, these truncated structures may not correctly reflect the quaternary structure of AChE in its true biological context. Murine and hAChE both exist as tetramers in their native forms and are anchored to the cellular membrane. By analogy, binding accessibility and the overall dynamics must be substantially different and significant in vivo, when compared to what is observed in currently available structures of the tetrameric form of BChE. New, even more sophisticated MD simulations may yield a more thorough structural analysis for the full‐length hAChE by providing new information on the allosteric mechanism and the resultant allostery causing the gorge to allow access to the active site (de Boer et al., 2021).

6. SUMMARY AND OUTLOOK

OP compounds rapidly inhibit AChE, which leads to irreversible damage to the nervous system if not treated rapidly. Crystal structures of AChE/OP complexes with nerve agents and insecticides have revealed detailed interactions between the active site and the covalently bound OP compounds. The general trends in these structures show that the phosphyl oxygen makes three hydrogen bonds with the three main‐chain amides of the three glycine residues in the oxyanion hole. The other typically larger phosphyl substituents interact with the choline binding pocket, while the smaller phosphyl or phosphonyl groups interact with the acyl binding pocket since the acyl binding pocket can accommodate smaller substituents such as methyl or ethoxy groups without significant distortion. For OPs with larger phosphyl groups facing the acyl pocket, the acyl loop necessarily distorts under steric constraint to accommodate the substituent. The aging process usually occurs by O‐dealkylation of the phosphyl groups in the choline binding pocket. However, an exception was seen in crystal structures of non‐aged and aged FeP‐mAChE adducts, where aging through O‐dealkylation of the ethoxy substituent facing the acyl binding site did not occur, but the isopropyl amino group facing the choline binding site prior to aging rotated to the acyl site after aging.

AChE is one of the most catalytically efficient enzymes known. This characteristic was thought to be attributed to its highly evolved active site where the geometry and electrostatic properties fit ACh perfectly. Thus, the catalytic activity occurred without the need for any conformational adjustments (Gerlits et al., 2021). This argument was supported by over 100 crystal structures of AChE that had been complexed with different OP inhibitors in many forms, including quasi‐reversible or reversible inhibitors and oxime reactivators, in both aged and non‐aged states, and at both cryogenic and room temperatures. The conformation of AChE's catalytic center was surprisingly conserved across all of these crystal structures. Nevertheless, AChE's high efficiency appears to be in conflict with the structural architecture of the enzyme, where the acetylcholine substrate is nearly equal in size to the width of the narrowest section of the long 20 Å gorge that must be traversed leading to the catalytic center. MD simulations reveal a highly dynamic enzyme where the catalytic gorge undergoes a ‘breathing’ motion as the gorge tunnel enlarges and reduces periodically in multi‐nanosecond intervals. Side‐chain movements open back and side doors on the bottom and the side of the gorge to allow reaction products and ligands to traverse through the catalytic site more efficiently than the static crystal structures depict. The PAS and the Ω‐loop, while exhibiting little conformational variation in the AChE crystal structures, are in fact dynamically involved with the gorge motion and substrate transport as revealed by MD studies.

Experimental evidence is still required to support the computationally predicted dynamic properties for AChE as clearly observed in MD simulations. Unfortunately, crystal structures, representing only steady‐state snapshots of this enzyme, are unlikely to provide additional information or a more detailed understanding of the extraordinary dynamic nature of this enzyme. Time‐resolved techniques optimized for examining slow dynamics, such as X‐ray footprinting or time‐resolved crystallography, may provide the necessary additional evidence that will fully address and explain AChE's unique capabilities.

AUTHOR CONTRIBUTIONS

Li‐Wei Hung: Conceptualization; writing – original draft; writing – review and editing; formal analysis; data curation; investigation. Karissa Y. Sanbonmatsu: Writing – review and editing. Robert F. Williams: Writing – review and editing. Julian C.‐H. Chen: Conceptualization; funding acquisition; writing – original draft; writing – review and editing; visualization; supervision; project administration.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Supporting information

TABLE S1. List of AChE structures in complex with OP compounds.

PRO-34-e70297-s001.xlsx (15.5KB, xlsx)

ACKNOWLEDGMENTS

This study was supported by the Laboratory Directed Research and Development program of Los Alamos National Laboratory under project number LDRD 20240384ER.

Hung L‐W, Sanbonmatsu KY, Williams RF, Chen JC‐H. Acetylcholinesterase: Structure, dynamics, and interactions with organophosphorus compounds. Protein Science. 2025;34(10):e70297. 10.1002/pro.70297

Review Editor: Lynn Kamerlin

Contributor Information

Li‐Wei Hung, Email: lwhung@lanl.gov.

Julian C.‐H. Chen, Email: chen_j@lanl.gov.

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

TABLE S1. List of AChE structures in complex with OP compounds.

PRO-34-e70297-s001.xlsx (15.5KB, xlsx)

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

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