Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase

Bucht, Gösta; Häggström, Britta; Radić, Zoran; Osterman, A.L.; Hjalmarsson, Karin

doi:10.1016/0167-4838(94)90195-3

Cited by 10 publications

(4 citation statements)

References 26 publications

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“…While there are no indications from crystallography of mobility of the homologous loop in AChE (36), flexibility of such a loop may be required for efficient catalysis. Substitutions of two aspartates at positions 92 and 93 in Torpedo californica AChE (equivalent to Asp94 and Asp95 in mouse AChE) with neutral residues at the base of this loop yield inactive enzyme (37) and are expected to break at least two salt bridges.…”

Section: Fasciculin-acetylcholinesterase Interactionsmentioning

confidence: 99%

Allosteric Control of Acetylcholinesterase Catalysis by Fasciculin

Radić

Quinn

Vellom

et al. 1995

Journal of Biological Chemistry

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110

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The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp86 is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp86 substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp86, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp86 which lies at the gorge base.

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Section: Fasciculin-acetylcholinesterase Interactionsmentioning

confidence: 99%

Allosteric Control of Acetylcholinesterase Catalysis by Fasciculin

Radić

Quinn

Vellom

et al. 1995

Journal of Biological Chemistry

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110

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“…The CAS is located at the bottom of the gorge, which has the catalytic triad, Ser203, His447 and Glu334 (Bourne et al 1995;Sussman et al 1991). There are also four other subsites near the CAS, which are the esteratic site (Pavlic 1975;Wilson 1951), oxyanion hole (Ordentlich et al 1998), anionic subsite (Bucht et al 1994) and the acyl pocket (Taylor and Radic 1994). The PAS (Asp74, Tyr124, Ser125, Trp286, Tyr337 and Tyr341) is located near the opening of the gorge, which is believed to attract and facilitate ACh movement into the gorge (Massoulié et al 1993;Sussman et al 1991).…”

Section: Phase II Reactionsmentioning

confidence: 99%

Evaluation of novel carbamate insecticides for neurotoxicity to non-target species

Jiang

Swale

Carlier

et al. 2013

Pesticide Biochemistry and Physiology

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Malaria (vector: Anopheles gambiae) is a major infectious disease that kills about 1 million people each year. For the improvement of its treatment and vector control during the past decades, several issues such as high medicine cost, insecticide resistance, and lack of an effective vaccine have prevented adequate control of malaria. Additionally, the low selectivity of malaria vector insecticides also presents a public health problem. The purpose of developing novel carbamate insecticides in our laboratory is to offer effective and selective insecticide options to achieve the ultimate goal of malaria control.First, 50% inhibition concentration (IC 50 ) data was collected from three mammalian AChEs with eight commercial carbamate insecticides by using the Ellman assay. The IC 50 values varied from 57 nM to 7358 nM. The AChE sensitivity pattern and level were shown to be similar between the recombinant mouse and ICR male mouse brain cortex homogenate (slope = 0.99, R 2 = 0.96).Then eight novel carbamate insecticides that are possible malaria vector control agents were selected for further neurotoxicity testing in non-target organisms. Neurotoxic esterase (NTE) assay showed that the novel carbamates did not significantly inhibit NTE, inhibition of which underlies a significant hazard for anticholinesterases, especially organophosphates, in several nontarget vertebrate organisms. The NTE activity in the presence of novel carbamate insecticides ranged from 93% to 116% of the control, while in the commercial group, bendiocarb significantly inhibited NTE, leaving only 76.5% of the initial reactivity at 1 mM inhibitor concentration.Further in vivo bioassay using Daphnia magna was conducted to compare the aquatic toxicity of commercial and novel carbamates. The data showed that except for PRC331 (3-tertbutylphenylmethylcarbamate), all novel carbamates were of similar potency as bendiocarb (LC 50 = 611 nM) for aquatic toxicity, and their LC 50 values ranged from 172 nM (PRC331) to 1109 nM.In conclusion, the novel carbamate insecticides would appear to be an improvement over commercial carbamate insecticides because of greater selectivity, negligible NTE inhibition capacity, but in some cases with potent in vivo toxicity to Daphnia magna. However, since the envisioned usage of these compounds is in bednets or as indoor residual sprays (IRS), any environmental exposures to nontarget aquatic organisms are expected to be minimal.iv ACKNOWLEDGEMENTS

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“…Their contribution to the catalysis cannot be studied by sitedirected mutagenesis. The replacement of a number of negatively charged residues Glu82, Glu278, Glu285, Asp342, Glu351, Glu382, Asp383 [44], Asp93 [45] and Asp131 [46] by neutral amino acids does not significantly change the activity of the enzyme towards the natural substrate. Only the mutant enzymes with Glu82 à Gln or Glu278 à Ala [44] mutations show twofold higher Km values in ASCh hydrolysis.…”

Section: Structure Of the Active Site Of The Enzyme And Amino Acid Rementioning

confidence: 99%

Acetylcholinesterase: Mechanism of Catalysis and Inhibition

Tõugu

2001

CMCCNSA

150

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Recent advances in the study of the catalytic properties of acetylcholinesterases have been reviewed. The main biological function of this enzyme is the fast termination of impulse transmission at cholinegric synapses by rapid hydrolysis of the neurotransmitter acetylcholine. Acetylcholinesterase has been often characterized as a perfect enzyme because its catalytic properties have been tuned to the highest possible limit. However, it seems paradoxical that the active site of this enzyme is buried deeply inside the enzyme molecule in the bottom of a narrow gorge restricting the traffic of substrates and products. The analysis of recent advances in enzymology and data on cholinesterase revealed rationality of this organization. The primary task of an enzyme catalyst is to lower the activation barrier for the chemical transformation of the substrate, and the catalytic power of the enzyme originates in polar solvation of the transition state by properly oriented dipoles inside the enzyme molecule. The active site gorge of acetylcholinesterase, containing multiple potential substrate binding areas, is responsible for "trapping" and delivery of substrate molecules to the active site. The phenomena of "allosteric modulation" and substrate inhibition arise as secondary effects of the presence of the narrow gorge lined with hydrophobic residues.

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Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase

Cited by 10 publications

References 26 publications

Allosteric Control of Acetylcholinesterase Catalysis by Fasciculin

Allosteric Control of Acetylcholinesterase Catalysis by Fasciculin

Evaluation of novel carbamate insecticides for neurotoxicity to non-target species

Acetylcholinesterase: Mechanism of Catalysis and Inhibition

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