The number of catalytic sites in acetylcholinesterase

By using two methods of titration, the number of active sites in acetylcholinesterase was determined. Either stepwise inhibition of the enzyme by an irreversible inhibitor, namely di-isopropyl phosphorofluoridate, or direct measurement of the concentration of active sites by titration with o-nitrophenyl dimethylcarbamate yielded an equivalent weight of approx. 130000 for an active site in acetylcholinesterase. This indicates two sites per molecule, since the native enzyme has a molecular weight of 260000.     

Modification of acetylcholinesterase with the fluorescent thiol reagent S-mercuric-N-dansylcysteine

11s acetylcholinesterase (EC 3.1.1.7) from Torpedo californica electroplax, purified by a combination of affinity and gel chromatography was found to react stoichiometrically with S-mercuric-N-dansylcysteine. Approximately four mols of reagent per mol of enzyme were incorporated when the modification was carried out in 1.0 mM Tris-C1, pH 7.5, either in the presence or absence of 0.1 M NaCl. Prior incubation of the enzyme with 1.0 x 10(-4) M Zn2+ allowed the incorporation of about six mols of reagent per mol of enzyme. Binding of the reagent produced shifts in the emission and excitation wavelength maxima which were similar for all reaction conditions; however the enhancement of fluorescence intensity which accompanied binding of reagent was dependent on the ionic composition of the reaction medium. The modified enzyme remained active towards the active site titrant 7-(dimethylcarbamoyloxy)-N-methylquinolinium and retained its sensitivity towards inactivation by Zn2+. The results suggest that acetylcholinesterase as prepared contains several accessible thiol groups, and that the bound reagent may prove to be a useful probe of ligand-induced conformational changes in acetylcholinesterase.     

Gallamine and tubocurarine as possible allosteric modifiers of soluble acetylcholinesterase activity at physiological ionic strength

Esters of dimethylcarbamic acid are known to be poor substrates of acetylcholinesterase. They carbamylate the active catalytic site of the enzyme and the subsequent decarbamylation is a slow but measurable process. Similarly, acetylcholinesterase can be phosphonylated, and the dephosphonylation is extremely slow. Rapid hydrolysis of phosphonylated acetylcholinesterase can be brought about by oximes, but dealkylation of the phosphonyl group on the enzyme (known as ageing) renders the inhibited enzyme insensitive to oximes.

In the present work, decarbamylation of dimethylcarbamyl-acetylcholinesterase and ageing of isopropylmethylphosphonyl-acetylcholinesterase were studied at a physiological ionic strength (154 mM). Gallamine, d-tubocurarine and alcuronium accelerated reactivation of dimethylcarbamyl-acetylcholinesterase. Gallamine and tubocurarine enhanced the effect of the nucleophile 3,3-dimethyl-1-butanol on decarbamylation, and the interaction was synergistic in the case of gallamine. Gallamine and tubocurarine retarded ageing of isopropylmethylphosphonyl-acetylcholinesterase, whereas 3,3-dimethyl-1-butanol had no effect. Nevertheless 3,3-dimethyl-1-butanol enhanced the retarding effects of gallamine and tubocurarine.

All these effects, except the effects of 3,3-dimethyl-1-butanol on ageing, had been previously observed at low ionic strength, in which case the effects were more marked and were observed at lower concentrations of the drugs. The effects at low ionic strength have been attributed to binding of the drugs to a peripheral site on the enzyme with a consequent change in conformation at the active site, leading to altered kinetic properties. The present results suggest that such allosteric effects may persist at physiological ionic strength. There have been few indications previously that this is so, particularly in the case of solubilised acetylcholinesterase.     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant k_on for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction. © 1998 John Wiley & Sons, Inc. Biopoly 46: 465–474, 1998     

Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate

Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20-fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol-anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.     

Acetylcholinesterase: diffusional encounter rate constants for dumbbell models of ligand.

For some enzymes, virtually every substrate molecule that encounters the entrance to the active site proceeds to reaction, at low substrate concentrations. Such diffusion-limited enzymes display high apparent bimolecular rate constants ((kcat/KM)), which depend strongly upon solvent viscosity. Some experimental studies provide evidence that acetylcholinesterase falls into this category. Interestingly, the asymmetric charge distribution of acetylcholinesterase, apparent from the crystallographic structure, suggests that its electrostatic field accelerates the encounter of its cationic substrate, acetylcholine, with the entrance to the active site. Here we report simulations of the diffusion of substrate in the electrostatic field of acetylcholinesterase. We find that the field indeed guides the substrate to the mouth of the active site. The computed encounter rate constants depend upon the particular relative geometries of substrate and enzyme that are considered to represent successful encounters. With loose reaction criteria, the computed rates exceed those measured experimentally, but the rate constants vary appropriately with ionic strength. Although more restrictive reaction criteria lower the computed rates, they also lead to unrealistic variation of the rate constants with ionic strength. That these simulations do not agree well with experiment suggests that the simple diffusion model is incomplete. Structural fluctuations in the enzyme or events after the encounter may well contribute to rate limitation.     

Amyloid-cholinesterase interactions. Implications for Alzheimer's disease

Acetylcholinesterase is an enzyme associated with senile plaques. Biochemical studies have indicated that acetylcholinesterase induces amyloid fibril formation by interaction throughout the peripherical anionic site of the enzyme forming highly toxic acetylcholinesterase-amyloid-beta peptide (Abeta) complexes. The pro-aggregating acetylcholinesterase effect is associated with the intrinsic amyloidogenic properties of the corresponding Abeta peptide. The neurotoxicity induced by acetylcholinesterase-Abeta complexes is higher than the that induced by the Abeta peptide alone, both in vitro and in vivo. The fact that acetylcholinesterase accelerates amyloid formation and the effect is sensitive to peripherical anionic site blockers of the enzyme, suggests that specific and new acetylcholinesterase inhibitors may well provide an attractive possibility for treating Alzheimer's disease. Recent studies also indicate that acetylcholinesterase induces the aggregation of prion protein with a similar dependence on the peripherical anionic site.     

Association of the synaptic form of acetylcholinesterase with extracellular matrix in cultured mouse muscle cells

Myotubes of a mouse muscle-cell line (C2) synthesize in culture a 16S form of acetylcholinesterase that is normally found only in regions of adult mouse muscle that contain endplates. The 16S enzyme in C2 cell extracts has the properties expected of acetylcholinesterase forms that have a collagen-like tail. In intact cells, the active site of the 16S acetylcholinesterase is protected by a membrane-impermeable inhibitor, and this form of the enzyme can be removed by treatment of the cells with collagenase. Thus the enzyme is extracellular. Its extraction by high ionic strength solutions lacking detergent suggests that the 16S form is associated with the extracellular matrix by ionic interactions. Histochemical staining shows focal patches of acetylcholinesterase activity on the cell surface. Collagenase treatment, which removes only the 16S form, abolishes this staining pattern, indicating that the patches consist of the 16S enzyme. We conclude that the 16S enzyme in C2 myotubes occurs in focal patches on the cell surface, where it is associated with the extracellular matrix.     

Progress in the development of enzyme-based nerve agent bioscavengers

Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers.     

Differences in conformational stability between native and phosphorylated acetylcholinesterase as evidenced by a monoclonal antibody.

Monoclonal antibody 25B1 generated against diisopropyl phosphorofluoridate inhibited fetal bovine serum acetylcholinesterase has been extensively characterized with respect to its anticholinesterase properties. This antibody demonstrated considerably different properties from previously reported inhibitory antibodies raised against acetylcholinesterase in terms of the degree of inhibition (greater than 98%), the high degree of specificity, and the stability of the antigen-antibody complex. Monoclonal antibody 25B1 appears to be directed against a conformational epitope located in close proximity to the catalytic center of the enzyme and was found to be most suitable for studying the stabilization of the active site of acetylcholinesterase against denaturation by heat or guanidine following phosphorylation by organophosphorus anticholinesterase compounds. This approach allowed the determination of stability rank order of various phosphorylated acetylcholinesterases. Among all the organophosphates tested, the combination of a methyl group and a negatively charged oxygen attached to the P atom, CH3P(O)(O-)-AChE, conferred the greatest protection to the active site of aged or nonaged organophosphoryl conjugates of acetylcholinesterase.     

Active-site catalytic efficiency of acetylcholinesterase molecular forms in Electrophorus, torpedo, rat and chicken

The active sites of acetylcholinesterase multiple forms from four widely different zoological species (Electrophorus, Torpedo, rat and chicken) were titrated using a stable, irreversible phosphorylating inhibitor (O-ethyl-S2-diisopropylaminoethyl methyl-phosphonothionate). In all cases, we found that within a given species, the molecular forms we examined were equivalent in their catalytic activity per active site. As pure preparations of the molecular forms of Electrophorus acetylcholinesterase were available, we were able to establish that one inhibitor molecule binds per monomer unit for each of them. This had already been shown by several authors for the tetrameric globular form, but not for the tailed molecules. Analysis of the phosphorylation reaction showed that they are equally reactive. Under our experimental conditions, their turnover number per site was 4.4 x 10(7) mol of acetylthiocholine hydrolysed . h-1 at 28 degrees C, pH 7.0. The corresponding value was less than half for Torpedo (1.64 x 10(7) mol . h-1), and again lower for rat (1.32 x 10(7) mol . h-1) and chicken (1.05 x 10(7) mol . h-1). In the case of rat acetylcholinesterase, the activity per active site of solubilized (with or without Triton X-100) and membrane-bound enzyme were identical. We discuss the implications of these findings with respect to the quaternary structure of acetylcholinesterase, and to the physico-chemical state and physiological properties of its molecular forms.     

Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge

A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH₂ residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k_cat) was increased while the Michaelis constant (K_m) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.     

Characterization of acetylcholinesterase in Caco-2 cells

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-beta-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.     

Acetylcholinesterase: Role of the enzyme's charge distribution in steering charged ligands toward the active site

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 Å radius centered in the enzyme monomer and separated from the protein surface by I-14 Å is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 Å radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within ±10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase. © 1996 John Wiley & Sons, Inc.     

Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

Identification of a modifier site on acetylcholinesterase with affinity for d-tubocurarine

Decamethonium and d-tubocurarine displace N-methylacridinium ion, a potent fluorescent inhibitor of acetylcholinesterase, from the surface of the enzyme. Decamethonium is competitive with N-methylacridinium which indicates that the binding sites for these ligands overlap. However, the displacement of N-methylacridinium ion by d-tubocurarine requires the existence of a binding site for d-tubocurarine in addition to the active site. Since the affinities for d-tubocurarine at both sites are comparable, two well defined ligand binding sites must exist for each catalytic site that is titratable by 7-dimethylcarbamyl-N-methylquinolinium iodide.     

Tetrameric mouse acetylcholinesterase: continuum diffusion rate calculations by solving the steady-state Smoluchowski equation using finite element methods

The tetramer is the most important form for acetylcholinesterase in physiological conditions, i.e., in the neuromuscular junction and the nervous system. It is important to study the diffusion of acetylcholine to the active sites of the tetrameric enzyme to understand the overall signal transduction process in these cellular components. Crystallographic studies revealed two different forms of tetramers, suggesting a flexible tetramer model for acetylcholinesterase. Using a recently developed finite element solver for the steady-state Smoluchowski equation, we have calculated the reaction rate for three mouse acetylcholinesterase tetramers using these two crystal structures and an intermediate structure as templates. Our results show that the reaction rates differ for different individual active sites in the compact tetramer crystal structure, and the rates are similar for different individual active sites in the other crystal structure and the intermediate structure. In the limit of zero salt, the reaction rates per active site for the tetramers are the same as that for the monomer, whereas at higher ionic strength, the rates per active site for the tetramers are approximately 67%-75% of the rate for the monomer. By analyzing the effect of electrostatic forces on ACh diffusion, we find that electrostatic forces play an even more important role for the tetramers than for the monomer. This study also shows that the finite element solver is well suited for solving the diffusion problem within complicated geometries.     

Inactivation of acetylcholinesterase with a bretylium tosylate photoaffinity probe

Azidobretylium tosylate (ABT), the p-azido analogue of bretylium tosylate, has been synthesized to serve as a photoaffinity probe for bretylium binding sites. Bretylium tosylate has antiarrhythmic action and also interacts with amiloride-sensitive sodium ion transport sites. Acetylcholinesterase was used as a model protein, and both bretylium and ABT are reversible inhibitors of this enzyme. The kinetic inhibition constants (Ki) were determined to be 40 microM for bretylium tosylate and 6 microM for ABT. The azido compound is photochemically labile and apparently irreversibly inactivates the enzyme. The rate was retarded by the addition of bretylium tosylate or 4-oxo-N,N,N-trimethylpentanaminium iodide (OTI). Sephadex G-25 chromatography further demonstrated the irreversible nature of the photoinactivation. Since ABT binds at or near the acetylcholinesterase active site, it may be a useful probe for the characterization of the enzyme active site.     

Allosteric Properties of Acetylcholinesterase

SEVERAL reports^1–3 have suggested that cholinergic ligands bind to acetylcholinesterase at sites distinct from the active centre. Changeux et al.⁴ demonstrated that acetylcholine binds to non-catalytic sites as well as to the active centre of the enzyme. Atropine arid 1-hyoscyamine have also been shown to interact with the enzyme at a region distinct from the active site^{5, 6}. We have therefore investigated whether acetylcholine and atropine are bound to the same site and whether this site is related to the acetylcholine receptor.     

Primary structures of the catalytic subunits from two molecular forms of acetylcholinesterase. A comparison of NH2-terminal and active center sequences

Two distinct classes of acetylcholinesterase exist in near equal amounts in the electric organ of Torpedo californica. A globular 5.6 S form is a dimer which possesses a hydrophobic region. The second form is present as elongated species that sediment at 17 and 13 S and contain structural subunits disulfide-linked to the catalytic subunits. Removal of the structural subunits by mild proteolysis yields a tetramer of catalytic subunits which sediments at 11 S. To compare the primary structures of the catalytic subunits of the 5.6 S and 11 S forms of acetylcholinesterase, amino acid sequences from the active sites and from the amino-terminal regions have been elucidated. Active site serines were labeled with [3H]isopropyl fluorophosphate. After digestion with trypsin, the resultant peptides were resolved by elution from a size-exclusion column followed by reverse-phase high performance liquid chromatography. Each active site tryptic peptide contained 24 residues and identical sequences were found in this peptide for the 5.6 S and 11 S forms of the enzyme. The sequence flanking the active site serine revealed extensive homology with the published sequence of human serum cholinesterase as well as a lesser degree of homology with other known serine proteases and esterases. The sequences of the amino-terminal region also appear to be identical for both enzyme forms although we note variation in the ratio of Glu and Gln at position 5. The amino-terminal sequence exhibits only partial homology with the published sequence of human serum cholinesterase.