Inhibitor Report for: Edrophonium

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

The active site of acetylcholinesterase is near the bottom of a long and narrow gorge. The dimensions of the gorge and the strong electrostatic field generated by the enzyme appear inconsistent with the enzyme's high turnover rate. Consequently, a "back door" mechanism involving movement of the reaction products through a transient opening near the active center was recently suggested. We investigated this hypothesis in human acetylcholinesterase by testing mutants at key residues (Glu-84, Trp-86, Asp-131, and Val-132) located near or along the putative back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by salt bridge Lys-132-Glu-452, are similar to that of the wild type enzyme. This indicates that the proposed back door is not a route for product clearance from the active site gorge of acetylcholinesterase and is probably of no functional relevance to its catalytic activity.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

Cholinesterases display a non-Michaelian behaviour with respect to substrate concentration. With the insect enzyme, there is an activation at low substrate concentrations and an inhibition at high concentrations. Previous studies allow us to propose a kinetic model involving a secondary non-productive binding site for the substrate. Unexpectedly, this secondary site has a very high affinity for the substrate when the enzyme is free. On the contrary, when the catalytic site of the enzyme is occupied a strong decrease of this affinity was observed. Moreover, a substrate molecule bound to the peripheral site results in a global decrease of the acylation and/or the deacylation step. Kinetic studies with three reversible inhibitors, tetramethylammonium, edrophonium and choline supported the kinetic model and enable its further refinement.

The potency of a series of anticholinesterase (anti-ChE) agents and serotonin-related amines as inhibitors of the aryl acylamidase (AAA) activity associated with electric eel acetylcholinesterase (AChE) (EC 3.1.1.7) and horse serum butyrylcholinesterase (BCHE) (EC 3.1.1.8) was examined and compared with the potency of the same compounds as ChE inhibitors. Neostigmine, physostigmine, BW 284C51, (+/-)-huperzine A, E2020, tacrine, edrophonium and heptyl-physostigmine were, in that order, the most potent in inhibiting eel AChE-associated AAA activity, their inhibitor constant (Ki) values being in the range 0.02-0.37 microM. The rank order of the same compounds as AChE inhibitors basically paralleled that of AAA, although they were in general stronger on AChE (Ki = 0.001-0.05). The peripheral anionic site inhibitors propidium and gallamine were inactive on AChE-associated AAA. Serotonin and its derivatives were slightly stronger on AAA (Ki = 7.5-30 microM) than on AChE (Ki = 20-140 microM). Tacrine (IC50 = 0.03 microM), diisopropylfluorophosphate (IC50 = 0.04 microM), heptyl-physostigmine (IC50 = 0.11 microM), physostigmine (IC50 = 0.15 microM) and tetra-iso-propylpyrophosphoramide (iso-OMPA) (IC50 = 0.75 microM) were the most potent in inhibiting horse serum BCHE-associated AAA activity. Serotonin and related amines were very weak on BCHE-associated AAA activity. These results indicate that the inhibitory potencies of the active site anti-ChE agents on the AAA activity associated with eel AChE and horse serum BCHE are closely correlated with their action on the respective ChE. In addition, the efficacy of tacrine, E2020, heptyl-physostigmine and (+/-)-huperzine A in the treatment of Alzheimer's disease is unlikely to be related to the action of these drugs on ChE-associated AAA.

BACKGROUND: Imbalance in cardiac sympathetic tone causes prolongation of the QTc interval of the ECG. On the other hand, impairment of the parasympathetic control of the heart rate caused by anticholinesterase-anticholinergic combinations might also affect the cardiac sympathetic tone and hence the QTc interval of the ECG. The main purpose of the present study was to compare the effects of four anticholinesterase-anticholinergic combinations used for the antagonism of the neuromuscular block on the QTc interval of the ECG, heart rate and arterial pressure. METHODS: Eighty-four ASA class I-II patients with a mean age of 32 to 37 yr undergoing otolaryngological surgery were randomly allocated to one of the following groups: neostigmine 40 microg/kg+glycopyrronium 8 microg/kg (Ne-Glyc), neostigmine 40 microg/kg+atropine 20 microg/kg (Ne-Atr), edrophonium 200 microg/kg+atropine 300 microg (Edr-Atr (1)), edrophonium 500 microg/kg+atropine 7 microg/kg (Edr-Atr (2)). QTc interval and heart rate were measured by a signal processing method based on an IBM/PC/xT-compatible microcomputer and arterial pressure with a sphygmomanometer at 1-min intervals up to 10 min after the injection of the drugs and immediately and 2 min after extubation. The ECG, lead II, was continuously recorded. Neuromuscular block was measured by a Datex relaxograph. RESULTS: In all groups, the most pronounced increase in both QTc interval, heart rate and arterial pressure occurred 1 min after the study drugs and immediately after extubation. In all groups, the mean QTc intervals at 1 and 2 min after the study drugs and after extubation were longer than the upper limit of the normal range (440 ms). Junctional rhythm occurred in 1 to 3 patients in all other groups with the exception of the Edr-Atr(1) group in which no cardiac arrhythmias occurred. At 1 min, the heart rate in the Ne-Atr group was at a significantly higher level than that in the Ne-Glyc group. From 3 to 6 min, the heart rate in the Edr-Atr(2) group and at 3 min in the Edr-Atr(1) group was at a lower level than the heart rate in the Ne-Glyc group. CONCLUSIONS: On the basis of the present results, anticholinesterase-anticholinergic combinations should be avoided in patients having a long QT interval syndrome or a prolonged QT interval from other causes. In addition, the cardiovascular stimulation caused by tracheal extubation should also be avoided in these patients.

Patients with syncope underwent head-up tilt testing at 60 degrees and 80 degrees followed by edrophonium or isoproterenol challenge when indicated. The 80 degrees tilt protocol and edrophonium provocation were found to be as effective or more effective in eliciting neurally mediated syncope in susceptible patients.

The bradycardia produced by neostigmine and edrophonium was examined according to its relation to cholinesterase inhibition and to its sensitivity to block by muscarinic receptor antagonists. For comparison, the ability of muscarinic antagonists to block the bradycardia produced by electrical stimulation of the vagus nerve was determined. METHODS: Cats were anaesthetized, vagotomized and propranolol-treated. Heart rate was continuously recorded. Erythrocyte cholinesterase activity of arterial blood was measured using a radiometric technique. The right vagus nerve was isolated for electrical stimulation. The muscarinic antagonists used were atropine, glycopyrrolate, pancuronium, gallamine, and AFDX-116. RESULTS: Neostigmine produced a dose-dependent decrease in cholinesterase activity which reached a plateau at a cumulative dose of 0.16 mg.kg-1 (ED50 0.009 +/- 0.003 mg.kg-1). Neostigmine produced a dose-dependent decrease in heart rate with the dose-response relationship (ED50 0.1 +/- 0.01 mg.kg-1; P = 0.0006) shifted to the right of that for the inhibition of cholinesterase activity. In contrast to the anticholinesterase effect, the bradycardic effect did not reach a plateau and continued to increase even at doses at which the cholinesterase inhibition was maximal. The maximal decrease in heart rate when the heart was still in sinus rhythm was by 81 +/- 13 bpm (49 +/- 7% of baseline), which was produced by a dose of 0.32 mg.kg-1. Edrophonium produced dose-dependent decreases in cholinesterase activity and heart rate, which were highly correlated (correlation coefficient r = 0.99, P < 0.0001). The ED50 of the reduction in heart rate (0.9 +/- 0.18 mg.kg-1) and cholinesterase activity (0.89 +/- 0.12 mg.kg-1) produced by edrophonium were similar. Moreover, the reduction in heart rate and cholinesterase activity produced by edrophonium reached a plateau at the same dose (6.4 mg.kg-1). At this dose, heart rate decreased by 22 +/- 2 bpm (14.6 +/- 0.9% of baseline). Compared to the bradycardia produced by stimulation of the vagus nerve, that produced by neostigmine was blocked by muscarinic antagonists at significantly lower doses while that produced by edrophonium was blocked at similar doses. CONCLUSIONS: The neostigmine-induced bradycardia is poorly correlated with cholinesterase inhibition compared to that produced by edrophonium, and has a higher sensitivity to muscarinic receptor antagonists compared to that produced by edrophonium or vagus nerve stimulation. These results are consistent with the hypothesis that the neostigmine-induced bradycardia is, in part, the result of neostigmine directly activating cholinergic receptors within the cardiac parasympathetic pathway. The bradycardia produced by edrophonium may be accounted for solely by an anticholinesterase action.

BACKGROUND: Reversal of neuromuscular blockade induced with pancuronium, d-tubocurarine, or doxacurium is achieved using smaller doses of neostigmine in adults than in children. Also, pancuronium- and doxacurium-induced blockade is reversed with smaller doses of edrophonium in children than in adults. The purpose of this study was to compare the spontaneous and neostigmine- and edrophonium-assisted recovery of mivacurium-induced neuromuscular block in adults and children. METHODS: Fifty-four adults, aged 40.1 +/- 10.9 yr, and 54 children, aged 4.9 +/- 0.7 yr, physical status ASA 1-2, were studied during propofol/fentanyl/nitrous oxide anesthesia. A Datex relaxograph was used to monitor the electromyographic response of the adductor pollicis to train-of-four stimulation of the ulnar nerve every 10 s. After induction of anesthesia, 0.2 mg x kg(-1) intravenous mivacurium was administered followed by an infusion to maintain 90-95% T1 block. At the end of surgery, one of four doses of neostigmine (5, 10, 20, and 50 micrograms x kg(-1)) or edrophonium (100, 200, 400, and 1,000 micrograms x kg(-1)) or placebo was given, by random allocation, when T1 had recovered to 10%. Values of T1 and train-of-four were measured for 10 min. RESULTS: Spontaneous recovery proceeded more rapidly in children than in adults. At 10 min, T1 had recovered to 97 +/- 2% (SD) in children compared with 69 +/- 11% in adults and train-of-four to 84 +/- 5% versus 30 +/- 13% (P<0.0001). In children, 10 min after reversal, recovery of T1 and train-of-four was not different from control after edrophonium and was enhanced only by the larger doses of neostigmine. In adults, recovery was accelerated by both edrophonium and neostigmine. Five minutes after reversal, recovery was improved by either drug in adults and in children. CONCLUSIONS: Spontaneous recovery from mivacurium- induced neuromuscular block is more rapid in children than in adults. Ten minutes after attempted reversal, recovery is accelerated by edrophonium and usually by neostigmine in adults but not in children. Thus, when reversal is required, edrophonium may be preferred to neostigmine.

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

Some anticholinesterase (anti-ChE) drugs induce airway smooth muscle contraction. Whether anti-ChE drugs stimulate muscarinic receptors in airway smooth muscle as well as nicotinic receptors in neuromuscular junction is unknown. Since there is a direct relationship between phosphatidylinositol (PI) response and airway smooth muscle contraction induced by muscarinic agonists, we examined the effects of neostigmine, physostigmine, pyridostigmine, and edrophonium on PI response in the airway smooth muscle. The rat tracheal slices were incubated in Krebs-Henseleit solution containing LiCl and [3H]myo-inositol in the presence of carbachol, anti-ChE, or none of them. [3H]inositol monophosphate (IP1), which is a degradation product of PI response, was counted with a liquid scintillation counter. Inositol monophosphate accumulation was stimulated by neostigmine, physostigmine, and pyridostigmine in a dose-dependent manner, but was not affected by edrophonium. These increases were completely inhibited by atropine. The results suggest that neostigmine, physostigmine, and pyridostigmine stimulate PI response in the airway smooth muscle, which would cause bronchoconstriction, while edrophonium does not affect PI response.

PURPOSE To examine the influence of anticholinesterase drugs neostigmine and edrophonium (which have different effects on plasma cholinesterase activity) administered for antagonism of neuromuscular block on the duration of action of mivacurium (a neuromuscular blocking drug metabolised by plasma cholinesterase). METHODS: This was a randomized study where mivacurium 0.15 mg.kg-1 was administered to a control group or after administration of neostigmine 40 micrograms.kg-1 or edrophonium 1 mg.kg-1 (n = 10 for each group) administered 10 min earlier for antagonism of atracurium-induced neuromuscular block. Neuromuscular block was measured by stimulation of the ulnar nerve in a train-of-four mode (TOF) and measuring the force of contraction of the adductor pollicis muscle. Baseline plasma cholinesterase activity was estimated before drug administration in all the groups and following anticholinesterase administration. RESULTS: The times to recovery of T1 (first response in the TOF) to 25 and 90% of control and of the TOF ratio to 0.7 after 0.15 mg.kg-1 of mivacurium were 47, 65 and 70 min in the neostigmine group; 25, 36 and 36 min in the edrophonium group and 17, 29 and 27 min respectively in the control group (P < 0.01). The plasma cholinesterase activity (PCHE) after neostigmine decreased from 6596 to 1959 U.L-1 (P < 0.001) but there was no change after edrophonium (6140 to 6396 U.L-1).
CONCLUSIONS: The duration of action of mivacurium is prolonged by previous administration of neostigmine and this is most likely to be due to inhibition of PCHE activity.

Three distinct acetylcholinesterases were detected in the annelid oligochaete Dendrobaena veneta. Two enzymes (alpha, beta), copurified from a Triton-X-100-soluble extract of whole animals by affinity (edrophonium-Sepharose) chromatography, were separately eluted from a Sephadex G-200 column. Gel-filtration chromatography, sedimentation analysis and SDS/PAGE showed the alpha and beta forms to be a globular dimer (110 kDa, 7.0 S) and a hydrophilic monomer (58 kDa, 5.0 S) respectively, both weakly linked to the cell membrane. The third form (gamma), also purified to homogeneity by slower filtration through an edrophonium-Sepharose matrix, proved to be an amphiphilic globular dimer (133 kDa, 7.0 S) with a phosphatidylinositol anchor giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. The alpha acetylcholinesterase showed a fairly low substrate specificity: the beta form hydrolyzed propionylthiocholine at the highest rate and was inactive on butyrylthiocholine; the gamma acetylcholinesterase, showing a marked active-site specificity with differently sized substrates, was likely functional in cholinergic synapses. Studies with inhibitors showed incomplete inhibition of all three acetylcholinesterase by 1 mM eserine and different sensitivity for edrophonium or procainamide. The alpha and beta forms, sensitive to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, were unaffected by tetra(monoisopropyl)-pyrophosphortetramide, while both these agents inhibited the gamma enzyme. All three forms showed excess-substrate inhibition by acetylthiocholine. Enzyme activity was histochemically localized in the nerve ring and its minor branches. Monomeric acetylcholinesterase (beta) is likely the only form present in the ganglionic glial framework.

The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed..

BACKGROUND Mivacurium, a nondepolarizing muscle relaxant, is metabolized by plasma cholinesterase. Although edrophonium does not alter plasma cholinesterase activity, we have observed that doses of edrophonium that antagonize paralysis from other nondepolarizing muscle relaxants are less effective with mivacurium. We speculated that edrophonium might after metabolism of mivacurium, thereby hindering antagonism of paralysis. Accordingly, we determined the effect of edrophonium on neuromuscular function and plasma mivacurium concentrations during constant mivacurium infusion. METHODS: We infused mivacurium to maintain 90% depression of adductor pollicis twitch tension and then gave edrophonium in doses ranging from 125-2,000 micrograms/kg without altering the mivacurium infusion. Peak twitch tension after edrophonium was determined to estimate the dose of edrophonium antagonizing 50% of twitch depression for antagonism of mivacurium; plasma cholinesterase activity and mivacurium concentrations before and after edrophonium were measured. Additional subjects were given 500 micrograms/kg edrophonium to antagonize continuous infusions of d-tubocurarine and vecuronium. RESULTS: With mivacurium, edrophonium increased twitch tension in a dose-dependent manner: the dose of edrophonium antagonizing 50% of twitch depression was 2,810 micrograms/kg. The largest dose of edrophonium (2,000 micrograms/kg) produced only 45 +/- 7% antagonism. Edrophonium, 500 micrograms/kg, antagonized mivacurium markedly less than it antagonized d-tubocurarine and vecuronium. Edrophonium increased plasma concentrations of the two potent stereoisomers of mivacurium 48% and 79%, these peaking at 1-2 min; plasma cholinesterase activity was unchanged. CONCLUSIONS: Edrophonium doses that antagonize d-tubocurarine and vecuronium are less effective in antagonizing the neuromuscular effects of mivacurium during constant infusion. Edrophonium increases plasma mivacurium concentrations, partly or completely explaining its limited efficacy; the mechanism by which edrophonium increases mivacurium concentrations remains unexplained. Our results demonstrate that antagonism of mivacurium by edrophonium is impaired, and therefore we question whether edrophonium should be used to antagonize mivacurium.

Rapid sequence induction of anaesthesia necessitating the use of suxamethonium may occasionally be needed soon after antagonism of neuromuscular block with anticholinesterase agents. The onset and duration of action of 1 mg kg-1 of suxamethonium was recorded in groups of 10 patients each, 5 or 10 min after the administration of edrophonium 1 mg kg-1 or neostigmine 40 micrograms kg-1 given for the antagonism of atracurium-induced neuromuscular block. Plasma cholinesterase activity was measured before, and 5 and 10 min after the administration of the anticholinesterases. A further 10 patients received suxamethonium 1 mg kg-1 without prior atracurium or anticholinesterase administration to serve as controls. The onset of action of suxamethonium was significantly prolonged when administered 5 min after both anticholinesterases, compared to the control group (P < 0.01). Recovery of suxamethonium block was delayed significantly after neostigmine, compared to both the edrophonium and the control groups (P < 0.05-0.001). Plasma cholinesterase activity was significantly reduced with the use of neostigmine but not with edrophonium (P < 0.001).

Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.

A 6-coumarin diazonium salt was synthesized and tested on Torpedo acetylcholinesterase as a site-directed irreversible probe for quaternary ammonium binding. The rate of the inactivation was examined as a function of time, inhibitor concentration, and pH, which allowed the determination of the dissociation and the rate constants of this efficient affinity labeling process. Protection experiments using tetramethylammonium, edrophonium, and propidium demonstrated that the labeling reaction occurred exclusively at the peripheral quaternary ammonium binding site of the enzyme. This result was confirmed by the modification of propidium binding at the peripheral site after inactivation reaction, as directly determined by fluorescence. Mutations of the likely labeled amino acid residues, Tyr70 and Tyr121, by histidine and phenylalanine indicated a predominant involvement of Tyr70 over Tyr121 in the coupling reaction.

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.

Pharmacokinetics of a very short-acting, a short-acting and two long-acting cholinesterase (ChE) inhibitors, edrophonium, neostigmine, pyridostigmine and ambenonium, respectively, were compared to elucidate the major determinant of their pharmacokinetics. No dose-dependency in pharmacokinetic behavior was observed within the range of 2-10 mumol/kg for edrophonium, 0.5-2 mumol/kg for pyridostigmine, 0.1-0.5 mumol/kg for neostigmine and 0.3-3 mumol/kg for ambenonium, respectively. Neostigmine has the shortest elimination half-life, and edrophonium, pyridostigmine and ambenonium follow in that. Four ChE inhibitors have similar Vdss values within the range of 0.3-0.7 l/kg, which is similar to the muscle/plasma concentration ratio of these drugs. The liver or kidney to plasma concentration ratio of all ChE inhibitors at 20min after i.v. administration ranged from 5 to 15. Small distribution volumes estimated from the plasma concentration profiles may reflect the distribution to muscle and to the extracellular space of other organs/tissues, while the rapid disappearance of ChE inhibitors from plasma may reflect the concentrative uptake to the liver and kidney.

Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.

Comparison of the effect of three 'peripheral' site ligands, propidium, d-tubocurarine, and gallamine, on acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) of Torpedo and chicken shows that all three are substantially more effective inhibitors of the Torpedo enzyme than of the chicken enzyme. In contrast, edrophonium, which is directed to the "anionic" subsite of the active site, inhibits the chicken and Torpedo enzymes equally effectively. Two bisquaternary ligands, decamethonium and 1,5-bis(4-allydimethylammoniumphenyl)pentan-3-one dibromide, which are believed to bridge the anionic subsite of the active site and the "peripheral" anionic site, are much weaker inhibitors of the chicken enzyme than of Torpedo acetylcholinesterase, whereas the shorter bisquaternary ligand hexamethonium inhibits the two enzymes similarly. The concentration dependence of activity towards the natural substrate acetylcholine is almost identical for the two enzymes, whereas substrate inhibition of chicken acetylcholinesterase is somewhat weaker than that of the Torpedo enzyme. The experimental data can be rationalized on the basis of the three-dimensional structure of the Torpedo enzyme and alignment of the chicken and Torpedo sequences; it is suggested that the absence, in the chicken enzyme, of two aromatic residues, Tyr-70 and Trp-279, that contribute to the peripheral site of Torpedo acetylcholinesterase is responsible for the differential effects of peripheral site ligands on the two enzymes.

The active site of acetylcholinesterase is near the bottom of a long and narrow gorge. The dimensions of the gorge and the strong electrostatic field generated by the enzyme appear inconsistent with the enzyme's high turnover rate. Consequently, a "back door" mechanism involving movement of the reaction products through a transient opening near the active center was recently suggested. We investigated this hypothesis in human acetylcholinesterase by testing mutants at key residues (Glu-84, Trp-86, Asp-131, and Val-132) located near or along the putative back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by salt bridge Lys-132-Glu-452, are similar to that of the wild type enzyme. This indicates that the proposed back door is not a route for product clearance from the active site gorge of acetylcholinesterase and is probably of no functional relevance to its catalytic activity.

Necator americanus (Nematoda: Strongyloidea), a human hookworm parasite, is known to release considerable amounts of acetylcholinesterase (AChE) [Pritchard, D. I., Leggett K. V., Rogan, M. T., McKean, P. G. & Brown, A. (1991) Necator americanus secretory acetylcholinesterase and its purification from excretory/secretory products by affinity chromatography, Parasite Immunol. 13, 187-199]. The present study deals with AChE activity recovered in sequential somatic extracts, and excretory/secretory products, of the adult stage of the parasite. 97% of AChE was extractable in low-salt and high-salt detergent-free buffers, and only 3% was solubilised by a further extraction in the presence of Triton X-100. AChE in all three extracts was affected by the AChE inhibitors eserine, bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide and edrophonium chloride, but was resistant to the effects of tetramonoisopropylpyrophosphortetramide, a butyrylcholinesterase inhibitor. Sucrose density centrifugation revealed that AChE in all somatic extracts (low-salt, high-salt and detergent) resolved almost exclusively as a single peak between 6.9-7.5 S, while excretory/secretory products resolved at 8.2 S. These values are all compatible with dimers of catalytic subunits and no evidence was found for the presence of higher oligomers such as asymmetric forms. The only sample to show a shift in sedimentation following the inclusion of detergent (Triton X-100, Brij 96) in the gradient was a component of the detergent-soluble extract, indicating the existence of a minor amphiphilic form. In low-salt-soluble and high-salt-soluble extracts, AChE was solubilised as a hydrophilic globular form, probably a dimeric G2. The analysis of diisopropylfluorophosphate-labelled extracts by SDS/PAGE, and unlabelled extracts by immunoblotting using a polyvalent antiserum to N. americanus AChE, indicated that the AChE isolated in each extract was biochemically and immunologically similar. The banding patterns obtained were comparable to that seen when purified AChE was analysed by SDS/PAGE and immunoblotted. This suggests that the basic catalytic subunit has a mass of 66-70 kDa with the active site being located in a 30-kDa domain. All experimental data indicate the existence of only one AChE class in Necator homologous to AChE of class B from Caenorhabditis elegans. The solubility characteristics and globular nature of this hookworm AChE suggest that its major function is as an excretory or secretory product. This again raises the question of the true biological function of this 'non-cholinergenic' nematode secretion.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.

A 35-year-old woman undergoing laparoscopic sterilisation developed prolonged apnoea after suxamethonium. Fresh frozen plasma was given to replenish plasma cholinesterase, but recovery of neuromuscular transmission was not accelerated. Routine use of quantitative neuromuscular monitoring simplified her postoperative management.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

To determine whether cholinesterase inhibition by an organophosphate would influence atracurium's neuromuscular blockade, six horses were anesthetized and paralyzed with atracurium (total of five injections per horse) on experimental Day 1, then were given trichlorfon (64 mg/kg per os) 6 days later. On Day 7, horses were anesthetized and paralyzed in the same manner as on experimental Day 1. Blood was taken to measure serum cholinesterase activity prior to anesthesia on Days 1 and 7. No significant difference was noted in atracurium's neuromuscular blocking activity between the 2 experimental days (P less than 0.05), despite Day 7 cholinesterase activity that was 16% of pre-trichlorfon values. For atracurium Injections 1 and 2-5, 85 and 43 micrograms/kg of atracurium, respectively, were required to produce a 95-99% reduction in hoof twitch. The time from injection to maximum twitch reduction was approximately 9 min after Injection 1 and 5 min after subsequent injections. Time from injection to maximum twitch reduction was significantly longer for Injection 1 than Injections 2-5 on both experimental days. The time from maximum twitch reduction until 10% recovery was approximately 8 min, with no significant difference between experimental days. The time for twitch recovery from 10 to 75% was approximately 17 min for all injections. Antagonism of atracurium with edrophonium caused the twitch height to return to pre-atracurium strength in approximately 7 min. Edrophonium caused a significant increase in arterial blood pressure. Heart rate change was variable after edrophonium

Stabilization of fetal bovine serum (FBS) acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AChE) and human butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) (BCHE) by ligands and inhibitors was studied as a function of physical and chemical perturbation. Denaturation of AChE occurred as a binary exponential function in the temperature range studied (50-56 degrees C); the slower fraction progressively diminished as the temperature was increased. Inclusion of ligands or inhibitors stabilized AChE as a function of temperature, ligand concentration and time. The rank order in which ligands stabilized AChE was: edrophonium greater than decamethonium greater than pralidoxime chloride much greater than procainamide. BCHE denaturation was retarded by ligands in the order: decamethonium greater than procainamide greater than edrophonium greater than pralidoxime. A plot of the quotient of the fast/slow ratio against the log of the 50% inhibitory concentration (I50) for ligands providing substantial protection yielded a linear relation, suggesting that these compounds stabilized AChE by a common mechanism involving the anionic site of the active center. Urea-induced cholinesterase denaturation was also retarded by these ligands.

The inhibitory effect of paraquat on cholinesterase activity was investigated in comparison with four paraquat derivatives, six monoquaternary ammoniums and six anticholinergic drugs. Inhibitor concentrations to cause 50% inhibition (I50) and Hill coefficients for three enzymes, human erythrocyte acetylcholinesterase (AChE), Electrophorus electricus AChE and human plasma butyrylcholinesterase (BCHE) were measured. The results obtained were as follows. The I50 for erythrocyte AChE was similar to the I50 for eel AChE. Secondary to edrophonium, diethylparaquat, paraquat, morfamquat and monoquat showed lower I50 for AChE, and possessed higher inhibition selectivity (IS), expressed as the ratio of I50 for BCHE to I50 for erythrocyte AChE. However, diquat showed higher I50 for AChE and lower IS, similar to the other monoquaternary ammoniums. A negative correlation was observed between log [I50 for erythrocyte AChE] and log [IS], among paraquat and its derivatives, monoquaternary ammoniums and anticholinergic drugs, respectively. With respect to Hill coefficients, these inhibitors could be classified into four groups, [1] competitive inhibitors: diquat, edrophonium, choline, tetramethylammonium and trimethylphenylammonium, [2] inhibitors showing negative cooperativity: paraquat, diethylparaquat, morfamquat, d-tubocurarine, atropine, gallamine and nicotine, [3] moderate type inhibitors: monoquat, hexamethonium and decamethonium. [4] the other type inhibitors showing positive cooperativity for erythrocyte AChE: tetraethylammonium and ethyltrimethylammonium.

The inhibition of human motor endplate cholinesterase by anticholinesterase compounds was studied using isolated muscle membrane preparation. Ambenonium was most potent, and edrophonium was least potent in inhibiting motor endplate cholinesterase. The slope of the regression line for inhibition of motor endplate cholinesterase was greatest for ambenonium, and smallest for neostigmine and edrophonium. These compounds were less potent inhibitors of plasma cholinesterase. Ambenonium was more specific, and other compounds were less specific inhibitors of motor endplate cholinesterase. In myasthenic patients, these compounds produced adequate inhibition of motor endplate cholinesterase even in the presence of relatively mild plasma cholinesterase inhibition.

A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.