Mutations acetylcholinesterase

Kolson, D.L. and Russell, R.L. (1985) A novel class of acetylcholinesterase, revealed by mutations, in the nematode Caenorhabditis elegans. Journal of Neurogenetics 2, 93-110. 

Mutero, A., Pralavorio, M., Bride, J.M. and Fournier, D. (1994) Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase. Proceedings of the National Academy of Sciences USA 91,5922—5926. 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

Chen, M., Han, Z., Quiao, X., and Qu, M., Resistance mechanisms and associated mutations in acetylcholinesterases in Sitobion avenue (Fabricius), Pestic. Biochem. Physiol., 87,189, 2007... 

Zhu, K.Y., Lee, S.H., and Clark, J.M., A point mutation of acetylcholinesterase associated with azin-phosmethyl resistance and reduced fitness in Colorado potato beetle, Pestic. Biochem. Physiol., 55,100,1996. 

Kovarik, Z., Radic, Z., Berman, H.A., Taylor, P. (2007). Mutation of acetylcholinesterase to enhance oxime-assisted catalytic turnover of methylphosphonates. Toxicology 233 79-84. 

There appear to be several, if not many, possible mutations which can confer various types of resistant acetylcholinesterases. Genetic approaches must be exploited to clarify this situation, including the cloning and analysis of genes for resistant acetylcholinesterases from which the critical amino acid substitutions can be discerned. To date, this gene has been cloned and sequenced from one insect, Drosophila, which was found to possess the identical active site sequence found in human, horse, and Torpedo cholinesterases ( 571-... 

Reduced reproduction is probably the most important endpoint in ecotoxicological risk assessments, whereas in pest control, death or changes in behavior are the most important. We simply want to kill the pest or make it run away. Toxicity tests are often based on what we call surrogate endpoints. We measure the level of an enzyme and how its activity is increased (e.g., CYP1A1) or reduced (acetylcholinesterase), how a toxicant reduces the light of a phosphorescent bacterium, or how much a bacterium mutates. Such endpoints are not always intuitively relevant to human health or environmental quality, but much research is done in order to find easy and relevant endpoints other than the fundamental ones. 

Menozzi. R, Shi, M. A., Lougarre, A.. Tang, Z. H., and Fournier, D. (2004). Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila mektrtogaster populations. BMC Evoi Biol. 4,4. 

Masson, P., Fromeni, M.-T, Sorenson, R, C., Bartels. C. F., and Lockridge, O. (1994). Mutation HLs322Asn in human acetylcholinesterase does not alter electrophoretic and catalytic properties of the erythrocyte enzyme. Blond 83, 3003-3005. 

Kovarik, Z Radic, Z., Berman, H, A., Simeon-Rudolf, V Reiner, E.. and Taylor, P, (2003). Acetylcholinesterase active centre and gorge conformations analysed hy combination mutations and enantiomeric phosphonates, Biochem.. f. 373, 33—40. 

Ohno, K-, Brcngman, L M., Tsujino, A., and Engd, A, G. (1998). Human endplate acetylcholinesterase deficiency caused by mutations in the collageii-like tail subunit (ColQ) of the asymmetric enzyme. Proc. Natl. Acad. Sci. USA 95,96,54-9659. 

Shi M. A., Lougarre A., Alies C., Fremaux L, Tang Z. H., Stojan J., and Fournier D., Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance, BMC. Evol. Biol., 6, 45, 2004. See http //www.biomedcentral.com/ content/pdf/1471-2148-4-45.pdf... 

Anatoxin-a(S) can therefore be measured by its inhibition of acetylcholinesterase whose enzyme activity can be measured by several ways. One example is its degradation of the acetylcholine analog, acetylthiocholine, and subsequent measurement of the released thiocholine by the sulfur reacting chemical Ellman s reagent. Acetylcholinesterase has been cloned and its mutation can increase the enzyme s sensitivity for anatoxin-a(S). Combining different acetylcholinesterase mutants with divergent specificity for anatoxin-a(S) and the above-mentioned organophosphate insecticides has enabled better analyte discrimination. This multiple enzyme method has been implemented in a biosensor that carries several of the acetylcholinesterase mutants. 

In Brownian dynamics simulations, electrostatic steering of the substrate toward the enzyme s active site(s) is generally, observed to increase the association rate over that for an uncharged model system without electrostatic interactions by one to two orders of magnitude. This increase is primarily due to electrostatic complementarity between the substrate and the enzyme active site (and access channel, if applicable). Thus rate acceleration can even be achieved when both the enzyme and the substrate have net charges of the same sign. This is also true when the rates are mostly rather insensitive to point mutations in the enzyme that are far from the active site (see e.g., simulations of acetylcholinesterase mutants ). 

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