Acetylcholinesterase: theory of noncompetitive inhibition

The theory of noncompetitive inhibition of acetylcholinesterase based on the binding of inhibitor to the acetylenzyme and the free enzyme was proven correct by demonstrating that tripropylammonium ion increases the steady-state concentration of acetylenzyme, as predicted by the theory. By contrast, the traditional theory that the inhibitor binds to the enzyme-substrate complex and the free enzyme predicts that the amount of acetylenzyme will be drastically reduced when the inhibition is high. A third theory involving all three types of binding remains possible.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.     

Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate.

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.     

乙酰胆碱酯酶在蚕豆保卫细胞中集中分布

动物细胞中,乙酰胆碱酯酶负责把乙酰胆碱水解为胆碱和乙酸以起到终止信号的作用。蚕豆（ＶｉｃｉａｆａｂａＬ．）保卫细胞原生质体表面具有特异的水解乙酰胆碱的酯酶,光可以激活该酶的活性。组织学定位的结果显示,酶反应产物主要分布于气孔保卫细胞内壁和腹壁的外侧及内壁和腹壁中,表明一种类似于动物突触传递的机制可能存在于气孔保卫细胞和周围细胞之间,即乙酰胆碱发挥作用后由乙酰胆碱酯酶分解以终止其作用;此外开放的气孔周围具有更高的酶活性,说明乙酰胆碱酯酶可通过水解气孔周围的乙酰胆碱而调控气孔运动     

Extensive Distribution of Acetylcholinesterase in Guard Cells of Vicia faba

Acetylcholinesterase, an enzyme responsible for hydrolyzing of acetylcholine to choline and acetic acid residues, is detected in the guard cell protoplasts. Extensive acetylcholinesterase activity has been found in the guard cell protoplasts as compared with the mesophyll cell protoplasts. Moreover, light could stimulate the enzyme activity. Localization of acetylcholinesterase in the stomata of Vicia faba L. was undertaken using Karnovsky and Roots cytochemical method. It was found that in the stomata of this plant products of acetylcholinesterase enzymatic reaction mainly appeared in the outer side of the guard cell ventral wall and inner wall. When the staining time was prolonged, products of acetylcholinesterase enzymatic reaction could also be found in the ventral and inner wall of the guard cells. In addition, more extensive product of enzymatic reaction was observed in the opened stomata than in the closed stomata. It was assumed that acetylcholineaterase may participate in the regulation of stomatal movement by hydrolyzing acetylcholine around the stomata.     

General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

On the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the reaction catalyzed by choline oxidase

Recent kinetic studies established that the positive charge on the trimethylammonium group of choline plays an important role in substrate binding and specificity in the reaction catalyzed by choline oxidase. In the present study, pH and solvent viscosity effects with the isosteric analogue of choline 3,3-dimethyl-butan-1-ol have been used to further dissect the contribution of the substrate positive charge to substrate binding and catalysis in the reaction catalyzed by choline oxidase. Both the kcat and kcat/Km values with 3,3-dimethyl-butan-1-ol increased to limiting values that were approximately 3- and approximately 400-times lower than those observed with choline, defining pKa values that were similar to the thermodynamic pKa value of approximately 7.5 previously determined. No effects of increased solvent viscosity were observed on the kcat and kcat/Km values with the substrate analogue at pH 8, suggesting that the chemical step of substrate oxidation is fully rate-limiting for the overall turnover and the reductive half-reaction in which the alcohol substrate is oxidized to the aldehyde. The kcat/Km value for oxygen determined with the substrate analogue was pH-independent in the pH range from 6 to 10, with an average value that was approximately 75-times lower than that previously determined with choline as substrate. These data are consistent with the positive charge headgroup of choline playing important roles for substrate binding and flavin oxidation, with minimal contribution to substrate oxidation.     

正交试验法确定测定意大利蜜蜂头部乙酰胆碱酯酶反应的最佳条件

用正交试验法研究了酶浓度、底物浓度、反应体系的pH值、反应温度和反应时间5个因素对测定意大利蜜蜂Apis mellifera ligustica Spinola头部乙酰胆碱酯酶(AChE)活性的影响, 并从试验组合中选出最佳条件。蜜蜂AChE活性的测定采用Gorun(1978)改进的Ellman方法, 以碘化硫代乙酰胆碱(ATCI)为底物, 5, 5' 二硫双硝基苯甲酸(DTNB)为显色剂, 测定反应物在412 nm波长下的光密度值, 用考马斯亮蓝G-250法测定蛋白质含量, 经计算得到蜜蜂头部AChE的比活力。对正交试验结果进行极差分析和方差分析, 结果表明各因素对实验结果影响的大小顺序为：温度＞pH值＞时间＞酶浓度＞底物浓度。并得出测定蜜蜂头部AChE活性的最佳条件是：酶终浓度0.2头/mL、底物终浓度0.8 mmol/L、pH值7.5、温度40℃及反应时间5 min。     

Gas phase ethyl acetate production in a batch bioreactor

Gas phase ethyl acetate production was studied using a porcine pancreatic lipase powder. It was observed that gaseous ethyl acetate was produced from gaseous ethanol and acetic acid. Accordingly, the effects of amount of lipase powder, gaseous ethanol and acetic acid concentrations, and reaction temperature on the performance of a batch bioreactor were investigated. Apparent Michaelis-Menten constant of ethanol was 0.163 [μM] and there was no inhibition by ethanol over the range investigated. As acetic acid concentration increased, ethyl acetate production increased to a maximum, then decreased, thus suggesting the inhibition effects by acetic acid. Over the reaction temperature of 25–55?°C, activation energy was calculated as 3.93 kcal/gmol and initial reaction rate was obtained as follows: r?=?75.7 exp(?1975.7/T) [μM/mg of lipase/hr]     

Substrate Specificity,Mode of Action,and of Inhibition by Organic Acids of Purified Acetylesterase from Sclerotinia Fungus

Acetylesterase (AcE) of Sclerotinia libertiana was purified approximately 1170-fold, and proved homogeneous by electrophoresis, ultracentrifugation and chromatography. The purified AcE hydrolyzed various acetyl esters in the following order; vinyl acetate, tri-acetin, n-butyl acetate, p-nitrophenylacetate, diacetin, ethylene glycol diacetate, monoacetin, ethyl acetate, acetylcholine, methyl acetate. It also had apparently a slight activity on tannic acid, benzoylcholine, methyl butyrate and acetic anhydride.

The mode of AcE reaction on these substrates could be divided into two types of group by Lineweaver-Burk plot, one forms the enzyme-substrate complex, ES, and the other, SES additionally combining substrate at a high substrate concentration.

From the inhibition experiment by organic acids, it was suggested that the neighbouring carboxyl groups of the di-, or tribasic acid such as citric, cis-aconitic, succinic, and maleic acid have a significance on inhibition of the AcE. Also, choline esterase inhibitor partially inhibited the activity on acetylcholine, and bivalent metal ion increased the activity on triacetin. Thus, the AcE was supposed to have a many adjacent sites of interaction with the substrate.     

A sensitive and regenerable biosensor for organophosphate pesticide based on self‐assembled multilayer film with CdTe as fluorescence probe

A fluorescence biosensor for organophosphorus pesticides was developed. A pH indicator, CdTe quantum dots, were used as an optical transducer of the inhibition of enzyme by analyte. Through the intervening agency of chitosan, the recognition elements (acetylcholinesterase and CdTe) were immobilized onto the surface of quartz by electrostatic attraction to form a self‐assembled multilayer film. In the absence of pesticide, acetylcholine was biocatalytically hydrolysed to yield acetic acid and choline. The released acid resulted in pH decrease, which was sensed by the immobilized pH indicator (CdTe). In the presence of pesticide, the action of acetylcholine was reduced; the fluorescence intensity of the film changed and was related to the concentration of pesticide. This multilayer film could be used as the biosensor for monocrotophos, with a detection limit of 3.20 × 10^?8 mol/L; the sensitivity was 100 times higher than that of CdTe in aqueous solution. The sensor was easily regenerated, and had good stability and selectivity for organophosphorus pesticides. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.     

THE EFFECT OF SPERMINE AND SPERMIDINE ON THE HYDROLYSIS OF ACETYLTHIOCHOLINE IN THE PRESENCE OF RAT CAUDATE NUCLEUS HOMOGENATE OR ACETYLCHOLINESTERASE FROM ELECTROPHORUS ELECTRICUS

Abstract— The effects of spermine and spermidine tetrahydrochloride on female Agus rat brain caudate nucleus homogenates, soluble acetylcholinesterase from the electric organ of Electrophorus electricus and acetylthiocholine iodide were studied. Measurements were made using an autoanalytical spectrophotometric method which measured the initial rate of reaction rapidly and accurately. Both polyamines interacted with the substrate, acetylthiocholine, causing an increase in the rate of its non-enzymatic hydrolysis. Slight inhibitory effects on acetylcholinesterase were also observed. Combined effect of the polyamine on the substrate and the enzyme showed an inhibition at low and activation at high (above 1 m m ) substrate concentrations.     

Determination of serum cholinesterase activity by liquid chromatography with electrochemical detection.

A sensitive enzymatic assay to measure cholinesterase activity in serum using liquid chromatography with electrochemical detection has been devised and used to examine cholinesterase inhibition in mice treated with diisopropyl phosphorofluoridate. Acetylcholine was used as substrate, and a postcolumn reactor containing immobilized choline oxidase converted the enzymatic product, choline, and the internal standard, ethylhomocholine, into the electrochemically active H2O2. The postcolumn reactor also contained acetylcholinesterase to allow the indirect detection of the substrate. Assay optimization included investigations of substrate concentration, buffer pH and ionic strength, enzyme concentration, incubation time, and reaction termination method. The optimized procedure is applicable to samples with activities of 0.11 to 269 mmol/ml/h. Intrasample coefficient of variation for mouse serum samples was 1.7% (n = 12), while intersample coefficient of variation was 8.0% (n = 5). The mean +/- SE serum cholinesterase activity found for controls and mice treated with diisopropyl phosphofluoridate (6.3 mg/kg, ip, 24 h prior) was 158.7 +/- 5.7 mumol/ml/h and 36.6 +/- 3.1 mumol/ml/h, respectively (P less than 0.001).     

Cholinesterases in peripheral nervous system. III. Validity of localisation around the node of Ranvier

Synopsis A series of experiments was designed to exclude the possibility that the acetylcholinesterase reaction of the rat's node of Ranvier is artefactual. Copper ions were not bound by perinodal acid mucopolysaccharides (or axons) at pH 6.0, which is the same value as that used in the histochemical cholinesterase techniques. No positive evidence was found for diffusion of acetylcholinesterase out of teased nerve fibres. Likewise, no evidence was obtained that acetylcholinesterase, acetylthiocholine or thiocholine are bound at pH 6.0 by perinodal acid mucopolysaccharides (or axons). It is concluded that local non-specific binding of copper, enzyme, substrate or reaction-product cannot account for the acetylcholinesterase reaction in either the node of Ranvier or the axon.     

The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.     

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.     

Two new methods monitoring kinetics of hydrolysis of acetylcholine and acetylthiocholine

Hydroxylamine and HPLC methods, measuring in vitro kinetics of enzymatic hydrolysis of acetylcholine or acetylthiocholine by cholinesterases, are described. The hydroxylamine method determines the dependence of substrate concentration vs. time, the HPLC method is able to measure simultaneously the time dependences of substrate and both primary products, choline or thiocholine, and acetic acid. Practical determinations are shown, comparison with known (above all Ellman's and pH-stat) methods, advantages and disadvantages are discussed.