Amino acids defining the acyl pocket of an invertebrate cholinesterase

Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in k_cat for BsCh; K_m and K_ss are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in k_cat for BsCh and a decrease in k_cat for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by π–π or cation–π interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.     

Cholinesterase inhibition in the bulb miteRhizoglyphus echinopus (Acari: Acaridae) in relation to the acaricidal action of organophosphates and carbamates

The 5000-g supernatant fraction of whole-bulb-mite homogenates was shown to possess a cholinesterase (ChE) that hydrolyzed acylcholine esters in the order acetyl>propionyl>butyryl. Acetyl--methylcholine, but not benzoylcholine, also was hydrolyzed as were acetylthiocholine and acetyl--methylthiocholine. No inhibition by excess substrate was observed at cholinester concentrations as high as 30mM. Cholinesterase activity was markedly insensitive to eserine and to certain other carbamates and organophosphates. Only organophosphates of the dimethylphosphate type generally were active ChE inhibitors. It was concluded that the inability of carbamates such as eserine, and organophosphates such as those with alkyl groups larger than dimethyl, to inhibit the bulb-mite ChE was probably a consequence of the nature of the esteratic site. The data suggested that ChE inhibition was likely involved in the toxicity to bulb mites of some of the toxic carbamates and organophosphates, but that it might not be the only mechanism involved, at least with several of the compounds.Contribution from the Missouri Agricultural Experiment Station, Columbia, MO, Journal Series No. 10 924.     

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.     

Design,synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors

A group of 2,4-disubstituted pyrimidine derivatives (7a–e, 8a–e and 9a–d) that possess a variety of C-2 aliphatic five- and six-membered heterocycloalkyl ring in conjunction with a C-4 arylalkylamino substituent were designed, synthesized and evaluated as cholinesterase (ChE) inhibitors. The steric and electronic properties at C-2 and C-4 positions of the pyrimidine ring were varied to investigate their effect on ChE inhibitory potency and selectivity. The structure–activity relationship (SAR) studies identified N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) as the most potent cholinesterase inhibitor (ChEI) with an IC₅₀ = 0.33 μM (acetylcholinesterase, AChE) and 2.30 μM (butyrylcholinesterase, BuChE). The molecular modeling studies indicate that within the AChE active site, the C-2 thiomorpholine substituent was oriented toward the cationic active site region (Trp84 and Phe330) whereas within the BuChE active site, it was oriented toward a hydrophobic region closer to the active site gorge entrance (Ala277). Accordingly, steric and electronic properties at the C-2 position of the pyrimidine ring play a critical role in ChE inhibition.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Inhibition of Ache: Structure-Activity Relationship Among Conformational Transition of Trp84 and Bimolecular Rate Constant

Abstract

In this study the authors attempt to correlate kinetic constants for carbamylation of AChE, by a series of carbamate inhibitors, with the conformational positioning of Trp84 in transition state complexes of the same carbamates with Torpedo AChE, as obtained by computerized molecular modelling. They present evidence for changes in the distance of the carbamates from the center of the indole ring which can be correlated with the bimolecular rate constants for inhibition. As a result the greater the distance from Trp84, the smaller the bimolecular inhibition constant value, k₁ (= k₂/K_a), becomes. In conclusion, the value of the biinolecular rate constant for selected AChE inhibitors (structural changes that have been hypothesised or natural alkaloids of unknown activity) which possess similar size and rigidity, can be obtained. Under these conditions energy minimization alone seems to be sufficient even to accurately predict protein-substrate interactions that actually occur. Modelling studies also suggest that conformational re-orientation of Trp84 in the transition state could produce an overall movement of the Cys67-Cys94 loop.     

Ultrastructural distribution of cholinesterase activity in the ventral nerve cord of the earthworm Lumbricus terrestris

Summary The fine structure and distribution of cholinesterase (ChE) activity in the ventral nerve cord of the earthworm (Lumbricus terrestris) was studied, using acetyl- and butyrylthiocholine iodides as substrates and iso-OMPA, 284C51 and eserine as inhibitors to discriminate between acetylcholinesterase (AChE) and other cholinesterase (ns.ChE) activities.The earthworm ventral nerve cord exhibits intense ChE activity. Both AChE and ns.ChE were present and they had identical distribution, being located mainly in the supportive glial cells. Most neurones of the ventral nerve cord contained no histochemically demonstrable activity. The ventral giant nerve cells were observed with the electron microscope to exhibit AChE activity. The enzyme was situated in the membranes of the rough-surfaced endoplasmic reticulum and in peculiar lamellated bodies but not in the membranes of the Golgi complex.     

Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.     

Characterization of a pseudocholinesterase purified from surgeonfish tissues confirms the atypical nature of this enzyme

The sialated, presumed-globular form of an atypical pseudocholinesterase (pseudo-ChE) previously described from surgeonfish tissues (Leibel: Comparative Biochemistry and Physiology 1988) has been purified to apparent homogeneity using a combination of salt fractionation along with ion-exchange and concanavalin A-Sepharose affinity chromatographic techniques. An overall 1,400-fold purification has been achieved with a 24% final yield of a cholinesterase (ChE) whose final specific activity is 50 mumol/min-mg. The purified enzyme was subjected to detailed biochemical and physical analysis. The purified pseudo-ChE is a sialated, globular, tetrameric enzyme with an apparent sedimentation coefficient of 11.5 S (+/- 0.5 S) and a molecular weight of 250 kilodaltons. The monomers are apparently not secured by disulfide bridges. The enzyme preferentially hydrolyzes acetyl(thio)choline but also hydrolyzes propionyl(thio)choline at reduced but comparable rates along with a wide variety of other noncholine esters. As such, it demonstrates the relative nonspecificity associated with classical pseudo-ChEs. However, the enzyme exhibits limited, but real, substrate inhibition with all choline esters as does true acetylcholinesterase (AChE). The enzyme is insensitive to the AChE inhibitor BW 284C51, sensitive to one (RO2-0683) of two (RO2-1250) pseudo-ChE inhibitors, and particularly sensitive to paraoxon inhibition (10(3)-10(4)-fold more so than AChE). It exhibits the short thermal half-life characteristic of pseudo-ChEs but not the expected ionic activation/inhibition profile. It is clear from this and other studies of atypical extrasynaptic cholinesterase activities occurring in other vertebrates that the orthodox categorization of cholinesterase as either "true" ("specific"; E.C. 3.1.1.7) or "pseudo" ("nonspecific"; E.C. 3.1.1.8) is inadequate to accommodate the increasing instances of ChE activities that exhibit atypical, intermediate properties.     

Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

The relationships between brain,serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Abstract

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

Structure of acetylcholinesterase complexed with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs.

BACKGROUND: Several cholinesterase inhibitors are either being utilized for symptomatic treatment of Alzheimer's disease or are in advanced clinical trials. E2020, marketed as Aricept, is a member of a large family of N-benzylpiperidine-based acetylcholinesterase (AChE) inhibitors developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies, prior to elucidation of the three-dimensional structure of Torpedo californica AChE (TcAChE). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. RESULTS: Our experimental structure of the E2020-TcAChE complex pinpoints specific interactions responsible for the high affinity and selectivity demonstrated previously. It shows that E2020 has a unique orientation along the active-site gorge, extending from the anionic subsite of the active site, at the bottom, to the peripheral anionic site, at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole', but only indirectly via solvent molecules. CONCLUSIONS: Our study shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.     

DEVELOPMENTAL CHANGES IN LEVELS AND FORMS OF CHOLINESTERASES IN MUSCLES OF NORMAL AND DYSTROPHIC CHICKENS

Abstract— Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were studied in vivo and during the first several months of development of pectoral and posterior latissimi dorsi (PLD) muscles in normal and dystrophic chickens. Muscle extracts were prepared in a high ionic strength-nonionic detergent medium in the presence of protease inhibitors, in order to obtain complete solubilization and to prevent degradation of intrinsic molecular forms of both enzymes. In both normal and dystrophic pectoral muscles levels of AChE and °ChE increase rapidly in vivo, °ChE accounting for 5–10% of total cholinesterase activity. In the normal pectoral muscle the concentration of both enzymes drops rapidly after hatching with increasing muscle mass; total AChE per muscle remains relatively constant for 30 days post-hatch. In the dystrophic pectoral muscle both AChE and °ChE accumulate after hatching, resulting in greatly elevated levels (approx 10–25-fold) of both enzymes throughout the period studied. Multiple molecular forms of AChE and °ChE are observed in the pectoral muscle by sucrose gradient centrifugation. Four principal forms are distinguished: two light (L₁, L₂), one medium (M), and one heavy (H₂). The °ChE forms are 0.5–1.0 S units lighter than the corresponding AChE forms. L₂ is the predominant light form of AChE, whereas L₁ is the major light °ChE form detected. The lighter forms of AChE predominate in normal and dystrophic embryonic pectoral muscle at day 14, being replaced by the H₂ form by day 19. H₂ is the major °ChE form detected at day 19. After hatching, H₂ AChE is the predominant form found in both of the normal muscles studied. In the dystrophic pectoral muscle, progressive accumulation of the L₂ form of AChE is detected as early as day 4 post-hatch; this form eventually becomes predominant, although the heavier forms are also elevated. In PLD muscle the same phenomenon occurs, but with a slower time course. In dystrophic pectoral muscle a similar rise in the L₁ form of °ChE is first observed by day 4, with heavier forms also elevated in the mature muscle. Thus the alteration in the control of these two enzymes in dystrophic fast-twitch muscles results in an accumulation of the light forms of AChE and °ChE.     

A comparative study of the interaction of the cholinesterase from the brain of the cabbage fly, the acetylcholinesterase from bovine erythrocytes and the butyrylcholinesterase from horse blood serum with organophosphorus inhibitors

Studies have been made of the effect of several organophosphorus inhibitors, R1(R2)P(O) . SCH2CH2SR and R1(R2)P(O)SCH2CH2SRR . -O4SCH3 (or -I), which differ by the structure of split (R, P) and phosphoryl (R1, R2) parts of the molecule, on cholinesterase (ChE) from the brain of the fly Delia brassicae, acetylcholinesterase (AChE) of the bovine erythrocytes and butyrylcholinesterase (BuChE) from the blood serum of the horse. For fly ChE, higher values of a constant (kII) of the inhibition rate (at pH 7.5 and temperature 25 degrees C) were obtained both with thiophosphates and with thiophosphonates. This finding reveals higher reactivity of the active centre of this enzyme, as well as significantly lower selectivity of the latter to the structure of organophosphorus inhibitors. The data obtained suggest the existence of differences in the size of hydrophobic regions of anionic and esterase parts of the active centre in ChE of the fly and AChE of mammals, as well as the existence of some similarity between ChE of the fly and BuChE.     

4-Aryl-4-oxo-N-phenyl-2-aminylbutyramides as acetyl- and butyrylcholinesterase inhibitors. Preparation,anticholinesterase activity,docking study,and 3D structure–activity relationship based on molecular interaction fields

Synthesis and anticholinesterase activity of 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, novel class of reversible, moderately potent cholinesterase inhibitors, are reported. Simple substituent variation on aroyl moiety changes anti-AChE activity for two orders of magnitude; also substitution and type of hetero(ali)cycle in position 2 of butanoic moiety govern AChE/BChE selectivity. The most potent compounds showed mixed-type inhibition, indicating their binding to free enzyme and enzyme–substrate complex. Alignment-independent 3D QSAR study on reported compounds, and compounds having similar potencies obtained from the literature, confirmed that alkyl substitution on aroyl moiety of molecules is requisite for inhibition activity. The presence of hydrophobic moiety at close distance from hydrogen bond acceptor has favorable influence on inhibition potency. Docking studies show that compounds probably bind in the middle of the AChE active site gorge, but are buried deeper inside BChE active site gorge, as a consequence of larger BChE gorge void.     

Acetylcholinesterase engineering for detection of insecticide residues

To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate.     

Multi-site inhibition of human plasma cholinesterase by cationic phenoxazine and phenothiazine dyes

Two cationic phenoxazine dyes, meldola blue (MB) and nile blue (NB), and the structurally related phenothiazine, methylene blue (MethB), were found to act as complex inhibitors of human plasma cholinesterase (butyrylcholinesterase, BChE). Studied at 25 degrees C, in 100mM MOPS buffer (pH 8.0), with butyrylthiocholine as substrate, the kinetic pattern of inhibition indicated cooperative I binding at 2 sites. Intrinsic K' values ( identical with[I](0.5)(2) extrapolated to [S]=0) for MB, NB and MethB were 0.64+/-0.05, 0.085+/-0.026 and 0.42+/-0.04 microM, respectively. Under the same experimental conditions the dyes acted as single-occupancy, hyperbolic-mixed inhibitors of electric eel acetylcholinesterase (AChE), with K(i)=0.035+/-0.010, 0.026+/-0.0034 and 0.017+/-0.0063 microM (for MB, NB, MethB); alpha (coefficient of competitive interaction)=1.8-2.4 and beta (coefficient of noncompetitive interaction)=0.15-0.28. The complexity of the BChE inhibitory effect of phenoxazine/phenothiazine dyes contrasted with that of conventional ChE inhibitors which cause single-occupancy (n=1), competitive or mixed inhibition in both AChE and BChE and signaled novel modes of ligand interaction at (or remote from) the active site gorge of the latter enzyme.     

Characterization of cholinesterase of muscularis muscle of Bufo marinus

We have characterized the cholinesterase (ChE) of muscularis muscle of Bufo marinus by selectively using specific inhibitors of acetylcholinesterase and pseudocholinesterase and observing susceptibility to inhibition when substrate is present in excess. The ChE activity in this preparation due to acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) was 90 and 10%, respectively. The optimum temperature and pH for the ChE were 38 degrees C and 7.4, respectively and the excess substrate inhibition was noted above a pS of 2.6. The Km for acetylthiocholine (ASCh) was 0.76 X 10(-4) M.     

Acyl Pocket of the Cholinesterase Active Center and Dialkylphosphates: Study of Interaction by Statistical Methods

The statistical analysis is performed of changes of the bimolecular rate constant value (log k _II) of inhibition of human AChE, mouse AChE, AChE of flies Musca domestica and Calliphora vicina, and horse BuChE by dialkylphosphates with the general formula (AlkO) ₂P(O)X at elongation of alkyl radicals and change of their branching in comparison with three physical-chemical characteristics (hydrophobicity, polarity, and volume of the side chain) of 6 amino acid residues in acyl and alkoxyl pockets variable in the studied ChE (Nos. 282, 287, 288, 290, 330, 335 in Torpedo ray AChE sequence). It has been shown that depending on structure of alkyl radicals, the rate of ChE interaction with OPI is determined by sterical hindrances to sorption (residues 282, 287, 290, 335), hydrophobic interactions (288) or polarity of microenvironment (287). This dependence in most cases is statistically significant; however, rather low values of the correlation coefficient indicate influence of structure of the OPI leaving part. The decrease of the statistical significance with elongation of alkyl radicals seems to be due to an increase of the number of possible conformational states of the OPI molecule.     

Partial purification and enzymatic characterization of acetylcholinesterase from the intertidal marine copepod Tigriopus brevicornis

Marine copepods such as Tigriopus brevicornis are widespread along the Atlantic coast of Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a subletal biomarker of exposure to neurotoxic supposes that ChE has been defined in copepods. This study reports the partial purification, separation and characterization of ChE extracted from T. brevicornis. This enzyme is apparently an acetylcholinesterase (AChE) since it hydrolyzed acetylthiocholine iodide at a higher rate than other substrates and was inhibited by eserine but not by iso-OMPA. Electrophoretic studies showed that there is probably a single dimeric form defined by its apparent molecular weight (200 kDa) and sensitivity to inhibitors. The kinetic properties of this ChE were compared with those of other invertebrate ChE.