7161-73-1Relevant articles and documents
Transition state structure and rate determination for the acylation stage of acetylcholinesterase catalyzed hydrolysis of (acetylthio)choline
Malany, Siobhan,Sawai, Monali,Sikorski, R. Steven,Seravalli, Javier,Quinn, Daniel M.,Radic, Zoran,Taylor, Palmer,Kronman, Chanoch,Velan, Baruch,Shafferman, Avigdor
, p. 2981 - 2987 (2000)
Rate-limiting steps and transition state structure for the acylation stage of acetylcholinesterase-catalyzed hydrolysis of (acetylthio)choline have been characterized by measuring substrate and solvent isotope effects and viscosity effects on the bimolecular rate constant k(E) (=k(cat)/K(m)). Substrate and solvent isotope effects have been measured for wild-type enzymes from Torpedo californica, human and mouse, and for various active site mutants of these enzymes. Sizable solvent isotope effects, (D2O)k(E) ~ 2, are observed when substrate β-deuterium isotope effects are most inverse, (βD)k(E) = 0.95; conversely, reactions that have (D2O)k(E) ~ 1 have substrate isotope effects of (βD)k(E) = 1.00. Proton inventories of k(E) provide a quantitative measure of the contributions by the successive steps, diffusional encounter of substrate with the active site and consequent chemical catalysis, to rate limitation of the acylation stage of catalysis. For reactions that have the largest solvent isotope effects and most inverse substrate isotope effects, proton inventories are linear or nearly so, consistent with prominent rate limitation by a chemical step whose transition state is stabilized by a single proton bridge. Reactions that have smaller solvent isotope effects and less inverse substrate isotope effects have nonlinear and upward bulging proton inventories, consistent with partial rate limitations by both diffusional encounter and chemical catalysis. Curve fitting of such proton inventories provides a measure of the commitment to catalysis that is in agreement with the effect of solvent viscosity on k(E) and with the results of a double isotope effect measurement, wherein (βD)k(E) is measured in both H2O and D2O. The results of these various experiments not only provide a model for the structure of the acylation transition state but also establish the validity of solvent isotope effects as a tool for quantitative characterization of rate limitation for acetylcholinesterase catalysis.
Purification and characterization of acetylcholinesterase from oriental fruit fly [Bactrocera dorsalis (Hendel)] (Diptera: Tephritidae)
Hsiao, Yi-Min,Lai, Jing-Ying,Liao, Hsiu-Ying,Feng, Hai-Tung
, p. 5340 - 5346 (2004)
An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the head of the insecticide susceptible oriental fruit fly, Bactrocera dorsalis (Hendel), by affinity chromatography of Triton X-100 extract. The degree of purification was about 8183-fold with recoveries of 52%. The molecular mass of purified AChE was 116 kDa for its native protein (nonreduced form) and 61 kDa for its subunits (reduced form) as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting that the homodimer of AChE linked with disulfide bonds. Nondenaturing PAGE of the purified AChE revealed only one molecular form. The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), propionylthiocholine, and S-butyrylthiocholine were 833.3, 222.2, and 57.5 μmol/min/mg, and the Michaelis constants (K m) were 87.9, 26.9, and 195.3 μM, respectively. More than 97% of AChE activity was inhibited by 10 μM eserine or BW284C51, but only 53% of the activity was inhibited by ethopropazine at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nevertheless, the purified AChE exhibited some distinctive characteristics including (i) a lack of the substrate inhibition phenomenon when using ATC as the hydrolyzing substrate and (ii) a higher Vm value for ATC than AChE from other insect species. These biochemical properties may show that AChE purified from the oriental fruit fly may have structural differences from those of other insect species.
Self-assembly of acetylcholinesterase on gold nanoparticles electrodeposited on graphite
Dimcheva, Nina,Horozova, Elena,Ivanov, Yavor,Godjevargova, Tzonka
, p. 1740 - 1748 (2013)
The immobilisation of AChE enzyme through chemisorption on Au-modified graphite was examined with view of its prospective application in the design of membraneless electrochemical biosensors for the assay of enzyme inhibitors. The developed immobilisation protocol has been based on a two-stage procedure, comprising i) electrodeposition of gold nanostructures on spectroscopic graphite; followed by ii) chemisorption of the enzyme onto gold nanoparticles. Both the coverage of the electrode surface with Au nanostructures and the conditions for enzyme immobilisation were optimised. The proposed electrode architecture together with the specific type of enzyme immobilisation allow for a long-term retaining of the enzyme catalytic activity. The extent of inhibition of the immobilised acetylcholinesterase enzyme by the organophosphorous compound monocrotophos has been found to depend linearly on its concentration over the range from 50 to 400 nmol mL-1 with sensitivity 77.2% inhibition per 1 μmol mL-1 of monocrotophos. [Figure not available: see fulltext.]
A fluorescence "turn-on" ensemble for acetylcholinesterase activity assay and inhibitor screening
Peng, Lihua,Zhang, Guanxin,Zhang, Deqing,Xiang, Junfeng,Zhao, Rui,Wang, Yilin,Zhu, Daoben
supporting information; experimental part, p. 4014 - 4017 (2009/12/06)
By making use of the aggregation-induced emission feature of compound 1 and the cascade reactions among acetylthiocholine iodide (ATC), AChE, and compound 2, a new fluorescence "turn-on" method is developed for AChE assay and inhibitor-screening.
