- Transition state structure and rate determination for the acylation stage of acetylcholinesterase catalyzed hydrolysis of (acetylthio)choline
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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.
- Malany, Siobhan,Sawai, Monali,Sikorski, R. Steven,Seravalli, Javier,Quinn, Daniel M.,Radic, Zoran,Taylor, Palmer,Kronman, Chanoch,Velan, Baruch,Shafferman, Avigdor
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- Structure-Guided Identification of a Small Molecule That Inhibits Anaerobic Choline Metabolism by Human Gut Bacteria
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The anaerobic gut microbial pathway that converts choline into trimethylamine (TMA) is broadly linked to human disease. Here, we describe the discovery that betaine aldehyde inhibits TMA production from choline by human gut bacterial isolates and a complex gut community. In vitro assays and a crystal structure suggest betaine aldehyde targets the gut microbial enzyme choline TMA-lyase (CutC). In our system, we do not observe activity for the previously reported CutC inhibitor 3,3-dimethylbutanol (DMB). The workflow we establish for identifying and characterizing betaine aldehyde provides a framework for developing additional inhibitors of gut microbial choline metabolism, including therapeutic candidates.
- Orman, Marina,Bodea, Smaranda,Funk, Michael A.,Campo, Ana Martínez-Del,Bollenbach, Maud,Drennan, Catherine L.,Balskus, Emily P.
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- Purification and characterization of acetylcholinesterase from oriental fruit fly [Bactrocera dorsalis (Hendel)] (Diptera: Tephritidae)
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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.
- Hsiao, Yi-Min,Lai, Jing-Ying,Liao, Hsiu-Ying,Feng, Hai-Tung
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- Functional Micellar Catalysis. Part 3. -Quantitative Analysis of the Catalitic Effects due to Functional Micelles and Comicelles
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The kinetic treatment of reactions catalysed by functional micelles and comicelles made up of chemically inert and functional surfactants, based on the pseudophase separation approach, allows the evaluation of the parameters defining the catalytic phenomena.The rate enhancements of activated ester hydrolysis due to cationic micelles of previously described functional surfactants, when compared to those due to analogous non-micellar model compounds, can be mainly accounted for by concentration and electrostatic effects.Desolvation and other medium effects on anionic micellar functions are not a relevant source of rate enhancement.The merits of comicellar solutions for the analysis under standard conditions of the catalytic effectiveness of a variety of functional surfactants are also presented.
- Fornasier, Roberto,Tonellato, Umberto
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- Self-assembly of acetylcholinesterase on gold nanoparticles electrodeposited on graphite
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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.]
- Dimcheva, Nina,Horozova, Elena,Ivanov, Yavor,Godjevargova, Tzonka
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- The kinetic inhibition of acetylcholinesterase from human herytrocyte by tacrine and some tacrine derivatives
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The kinetics of the reaction catalyzed by human erythrocyte Acetylcholinesterase (ACHE) is studied in the presence of its inhibitor Tacrine (1,2,3,4-tetrahydro-9-acridinamine), and of two newly synthesized compounds, 6-metoxy-Tacrine and N-eptyl-Tacrine. The proposed kinetic model describes the rate of the enzymatic reaction in terms of competitive and uncompetitive mixed inhibition of the three different inhibitors. The kinetic parameters describing the rate of the reaction are obtained. The competitive and uncompetitive inhibition constants of the different inhibitors are reported and the mixed competitive-uncompetitive inhibition is discussed.
- Porcelli, Fernando,Delfini, Maurizio,Del Giudice, Maria Rosaria
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- A fluorescence "turn-on" ensemble for acetylcholinesterase activity assay and inhibitor screening
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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.
- Peng, Lihua,Zhang, Guanxin,Zhang, Deqing,Xiang, Junfeng,Zhao, Rui,Wang, Yilin,Zhu, Daoben
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supporting information; experimental part
p. 4014 - 4017
(2009/12/06)
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- Design and activity of cationic fullerene derivatives as inhibitors of acetylcholinesterase
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Four different regioisomers of cationic bis-N,N- dimethylfulleropyrrolidinium salts have been prepared and evaluated as inhibitors of the enzymatic activity of acetylcholinesterase. These fullerene-based derivatives were found to be noncompetitive inhibitors of acetylthiocholine hydrolysis. Molecular modelling was used to describe the possible interactions between the fullerene cage and the amino acids surrounding the cavity of the enzyme. The cationic C60 derivatives used in this study represent a new class of molecules potentially able to modulate the enzymatic activity of acetylcholinesterase. The Royal Society of Chemistry 2006.
- Pastorin, Giorgia,Marchesan, Silvia,Hoebeke, Johan,Da Ros, Tatiana,Ehret-Sabatier, Laurence,Briand, Jean-Paul,Prato, Maurizio,Bianco, Alberto
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p. 2556 - 2562
(2008/09/21)
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- Delineation and decomposition of energies involved in quaternary ammonium binding in the active site of acetylcholinesterase
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The quaternary ammonium binding locus in the active site of mammalian acetylcholinesterase is subtended by the side chains of Trp86, Tyr133, Glu202, and Tyr337. Linear free-energy relationships define the interactions involved in molecular recognition by mouse acetylcholinesterase of the quaternary ammonium moiety of ligands. For substrates CH3C(=O)XCH2CH2Y [X = O, Y = CHMe2, or CH2CH3; X = S, Y = H, NH+Me2, or N+Me3] and trifluoroacetophenone transition state analogue inhibitors m-YC6H4C(=O)CF3 [Y = H, Me, Et, iPr, tBu, CF3, NH2, NO2, NMe2, or N+Me3], log(k(cat)/K(m)) and pK(i) depend linearly on the molar refractivity, but not the hydrophobicity, of the substituents Y. These correlations indicate that, in the acylation stage of catalysis, interactions in the quaternary ammonium binding locus stabilize the tetrahedral intermediate (as modeled by transition state analogue affinity) by (5 x 105)-fold (ΔΔG(TI) = -32.5 kJ mol-1) and the transition state by (2 x 104)-fold (ΔΔG(+) = -24.5 kJ mol-1). To evaluate the contribution of cation-π interactions, Trp86 was convened into Tyr, Phe, and Ala by site-specific mutagenesis. For this set of enzymes, a linear free-energy relationship is observed between the pK(i) values for inhibitions by the respective neutral and cationic transition state analogue inhibitors, m-tert-butyltrifluoroacetophenone and m-(N,N,N- trimethylammonio)-trifluoroacetophenone, which indicates that the free energy released on interaction of the quaternary, ammonium moiety with Trp86 arises about equally from cation-π and charge-independent interactions.
- Quinn, Daniel M.,Feaster, Shawn R.,Nair, Haridasan K.,Baker, Nathan A.,Radic, Zoran,Taylor, Palmer
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p. 2975 - 2980
(2007/10/03)
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- Combined effect of organophosphorus hydrolase and grime on the reactivation rate of diethylphosphoryl-acetylcholinesterase conjugates
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Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is 3 combined post exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl AChE (DEP-AChE) conjugates, obtained by the use of various diethylphosphates, were (determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 μg/mL blond would result in a 100 fold decrease in the concentration of circulating anti AChE pesticides within less than one blood circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP based pesticidcs, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium containing OPs. This indicates that hydrolysis of phosyhorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.
- Ashani, Yacov,Leader, Haim,Rothschild, Nathan,Dosoretz, Carlos
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p. 159 - 168
(2007/10/03)
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