Acetylcholinesterase stability

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],... 

A.K. Singh, A.W. Flounders, J.V. Volponi, C.S. Ashley, K. Wally, and J.S. Schoeniger, Development of sensors for direct detection of organophosphates. Part I immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports. Biosens. Bioelectron. 14, 703-713 (1999). 

Antibody 15C5 was able to catalyse the hydrolysis of the triester [105] with cat 2.65 x 10 3 min 1 whilst a second antibody from the same immunization programme was later found to hydrolyse the acetylcholinesterase inhibitor Paraoxon [106] with kcat = 1.95 x 10 3min-1 at 25°C (Appendix entry 6.2) (Lavey and Janda, 1996b). Antibody 3H5 showed Michaelis-Menten kinetics and was strongly inhibited by the hapten [104]. It exhibited a linear dependence of the rate of hydrolysis on hydroxide ion concentration, suggesting that 3H5 effects catalysis by transition state stabilization rather than by general acid/base catalysis. 

Enol esters are distinct from other esters not because of a particular stability or lability toward hydrolases, but due to their hydrolysis releasing a ghost alcohol (an enol), which may immediately tautomerize to the corresponding aldehyde or ketone. A well-studied example is that of vinyl acetate (CH3-C0-0-CH=CH2), a xenobiotic of great industrial importance that, upon hydrolysis, liberates acetic acid (CH3-CO-OH) and acetaldehyde (CH3-CHO), the stable tautomer of vinyl alcohol [25], The results of two studies are compiled in Table 7.1, and demonstrate that vinyl acetate is a very good substrate of carboxylesterase (EC 3.1.1.1) but not of acetylcholinesterase (EC 3.1.1.7) or cholinesterase (EC 3.1.1.8). The presence of carboxylesterase in rat plasma but not in human plasma explains the difference between these two preparations, although the different experimental conditions in the two studies make further interpretation difficult. 

The frequent occurrence of sialylated enzymes, or even of multiple forms, which are sometimes tissue-dependent, with a varying number of sialyl residues as, for example, in y-glutamyltranspeptida.se (EC 2.3.2.2),456,457 is not yet fully understood. Although the activity of most of these enzymes is not influenced by removal of sialic acid,454 the activity of monoamine oxidase A (EC 1.4.3.4) of outer mitochondrial membranes of rat liver has been shown to be destroyed by treatment with sialidase438 the substrate specificity of acetylcholinesterase (EC 3.1.1.7) is altered,459 the kinetic properties of human acid and alkaline phosphatases (EC 3.1.3.1 and 3.1.3.2) are changed, and the stability of a-D-galactosidase (EC 3.2.1.22) is drastically lowered.415 In these cases, an influence of sialyl residues on the conformation of the enzyme is assumed, but awaits firm evidence. 

It is important to know that the inhibition of acetylcholinesterase by OPs is through an attack on the relatively positive phosphorus atom by the hydroxyl group of a serine residue at the enzyme s site of action. Electron withdrawing substitutions within the OP tend to make the phosphorus more positive and, therefore, more reactive. Unfortunately, this type of substitution also makes the compound less stable hydrolytically. The discovery and development of OP insecticides has always been a balance between activity against the enzyme of the insect, selectivity in comparison with mammalian systems and stability within the insect. The binding of OPs to acetylcholinesterase is often irreversible. Typical OP insecticides are shown in Figure 3.3. 

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

Deprez, P., Doss-Pepe, E., Brodsky, B., and Inestrosa, N. C. (2000). Interaction of the collagen-like tail of asymmetric acetylcholinesterase with heparin depends on triple-helical conformation, sequence and stability. Biochem. J. 350, 283-290. 

S. Estrada-Mondaca and D. Fournier, Stabilization of recombinant Drosophila acetylcholinesterase, Prot. Exp. Purif., 12 (1998) 166-172. 

Our approach to this problem has been to synthesize several of these potential transformation products, to test their ability to inhibit acetylcholinesterase in vitro as well as their toxicity to a suitable insect indicator species and to begin an assessment of their stability under environmental conditions. In this connection it was deemed necessary to measure the volatility of the more potent inhibitors. 

Physostigmine is one of the major alkaloids in calabar bean. It has been shown to inhibit acetylcholinesterase at low concentration and to reverse the toxic effects resulting from diazepam overdose. The phytostigmine skeleton is easily obtained in very good yields from an appropriate non-stabilized imidate methylide.162,292,465... 

Martinez-Pena Y, Valenzuela I, Akaaboune M. 2007. Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice. Mol Biol Cell 18 2904-2911. 

Scorpionid secretions represent a mixture of neurotoxic polypeptide toxins, proteolytic and hemolytic enzymes (phospholipases A, acetylcholinesterases, ribonucleases, hyaluronidases), and biogenic amines (serotonin, tryptamine, histamine). The polypeptide toxins (the so-called scorpamines) contain fewer than 40 or 60-76 mostly alkaline and aromatic amino acids stabilized by four disulfide bridges.20 96... 

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