Acetylcholinesterase reaction

Figure 14.13 The kinetic sequence of reactions that control the cyclic AMP concentration, and its binding to the effector system, and the kinetic sequence that controls the concentration of a neurotransmitter and its binding to the receptor on the postsyn-aptic membrane. Processes (1) are reactions catalysed by adenyl cyclase, and exocytosis. Reactions (2) are catalysed by phosphodiesterase and, for example, acetylcholinesterase. Reactions (3) are the interactions between the messenger and the effector system both the latter are equilibrium binding processes. (See Chapter 12 (p. 266) for discussions of equilibrium binding.)...

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

Siojan, f.. Golicnik, M., and Fournier, D, (2004). Rational polynomial equation as an unbiased approach for the kinetic studies of Dm.sophila melanogasier acetylcholinesterase reaction mechanism. Bhxiiim. Biophy.s. Acta 1703, 53-61. 

Y. Cheng, J. K. Suen, D. Zhang, S. D. Bond, Y. Zhang, Y. Song, N. A. Baker, C. L. Baja], M. J. Holst, and J. A. McCammon. Finite element analysis of the time-dependent Smoluchowski equation for acetylcholinesterase reaction rate calculations. Biophys. J., 92(10) 3397-406, 2007. 

Bajaj, M. J. Holst, and J. A. McCammon, Biophys. J., 92,3397-3406 (2007). Finite Element Analysis of the Time-Dependent Smoluchowski Equation for Acetylcholinesterase Reaction Rate Calculations. 

Enzyme-Catalyzed Reactions Enzymes are highly specific catalysts for biochemical reactions, with each enzyme showing a selectivity for a single reactant, or substrate. For example, acetylcholinesterase is an enzyme that catalyzes the decomposition of the neurotransmitter acetylcholine to choline and acetic acid. Many enzyme-substrate reactions follow a simple mechanism consisting of the initial formation of an enzyme-substrate complex, ES, which subsequently decomposes to form product, releasing the enzyme to react again. 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Berkman CE, Ryu S, Quinn DA, et al. 1993. Kinetics of the postinhibitory reactions of acetylcholinesterase poisoned by chiral isomalathion A surprising nonreactivation induced by the Rp stererisomers. Chem Res Toxicol 6 28-32. 

Absorbance- and reflectance-based measurements are widespread, as there are many enzymatic reaction products or intermediates that are colored or if not, can react with the appropriate indicator. Sensors using acetylcholinesterase for carbamate pesticides detection are an example of indirect optical fiber biosensors. This enzyme catalyses the hydrolysis of acetylcholine with concomitant decrease in pH41 ... 

Observations The preliminary treatment of the cholinesterase-containing material with allelochemical (or other compound, e.g. active oxygen species, ozone free radicals and peroxides, formed in allelopathic relations) is for 30 min, then a substrate acetylcholinesterase is added to the reaction medium and final reaction of hydrolysis is for 1 h. 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

In this system, choline formed by acetylcholinesterase is oxidized by choline oxidase and the hydrogen peroxide produced is determined using the luminol/peroxidase CL reaction. The sensor has been used for the analysis of Paraoxon and Aldicarb pesticides, with detection limits of 0.75 pg/L and 4 pg/ L, respectively. Recoveries in the range of 81-108% in contaminated samples of soils and vegetables were obtained. 

Thioesters play a paramount biochemical role in the metabolism of fatty acids and lipids. Indeed, fatty acyl-coenzyme A thioesters are pivotal in fatty acid anabolism and catabolism, in protein acylation, and in the synthesis of triacylglycerols, phospholipids and cholesterol esters [145], It is in these reactions that the peculiar reactivity of thioesters is of such significance. Many hydrolases, and mainly mitochondrial thiolester hydrolases (EC 3.1.2), are able to cleave thioesters. In addition, cholinesterases and carboxylesterases show some activity, but this is not a constant property of these enzymes since, for example, carboxylesterases from human monocytes were found to be inactive toward some endogenous thioesters [35] [146], In contrast, allococaine benzoyl thioester was found to be a good substrate of pig liver esterase, human and mouse butyrylcholinesterase, and mouse acetylcholinesterase [147],... 

Phosphorothioates and phosphonothioates are of particular significance as insecticides. Schematically, it can be stated that these xenobiotics undergo activation by oxidative desulfuration, and detoxification by hydrolytic cleavage. Oxidative desulfuration transforms phosphorothioates and phosphonothioates to the corresponding oxon derivatives (see Chapt. 7 in [59]), which are highly toxic as potent inactivators of acetylcholinesterase [69]. This route of toxification can be competitive with and/or followed by cleavage reactions, which can be either hydrolytic or oxidative. 

Lewis, W. G. Green, L. G. Grynszpan, F. Radic, Z. Carlier, P. R. Taylor, R Finn, M. G. Sharpless, K. B. Click chemistry in situ Acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem. Int. Ed. 2002,41,1053-1057. 

Recent developments have led to agents with a built-in functional group that allows more rapid metabolism. Initially, the presence of ester groupings, as in suxamethonium, allowed fairly rapid metabolism in the body via esterase enzymes that hydrolyse these linkages. The enzyme involved appears to be a non-specific serum acetylcholinesterase (see Box 13.4). Even better is the inclusion of functionalities that allow additional degradation via an elimination reaction. Such an agent is atracurium. 

Tlie neurotransmitter acetylcholine is both a quaternary ammonium compound (see Box 6.7) and an ester. After interaction with its receptor, acetylcholine is normally degraded by hydrolysis in a reaction catalysed by the enzyme acetylcholinesterase. This enzyme contains a serine residue that acts as the nucleophile, hydrolysing the ester linkage in acetylcholine (see Box 13.4). This effectively acetylates the serine hydroxyl, and is an example of transesterification (see Section 7.9.1). For continuation of acetylcholine degradation, the original form of the enzyme must be regenerated by a further ester hydrolysis reaction. 

However, not included in the above mechanisms are other amino acid side-chains at the active site, whose special role will be to help bind the reagents in the required conformation for the reaction to occur. Examples of such interactions are found with acetylcholinesterase and chymotrypsin, representatives of a group of hydrolytic enzymes termed serine hydrolases, in that a specific serine amino acid residue is crucial for the mechanism of action. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

The reaction of a chemically active substance with a group located in an enzyme s active site, such that the stoichiometry of active sites can be determined analytically. See Acetylcholinesterase Affinity Labeling Diiso-propylfluorophosphate... 

A reaction whose rate is limited (or controlled) only by the speed with which reactants diffuse to each other. For a ligand binding to a protein, the bimolecular rate constant for diffusion-limited association is around 10 M s. The enzyme acetylcholinesterase has an apparent on-rate constant of 1.6 x 10 M s with its natural cationic substrate acetylcholine, and the on-rate constant of about 6 X 10 with acetylselenoylcholine and about... 

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