Involvement of Deacylation in Activation of Substrate Hydrolysis by Drosophila Acetylcholinesterase

Abstract

Insect acetylcholinesterase (AChE), an enzyme whose catalytic site is located at the bottom of a gorge-like structure, hydrolyzes its substrate over a wide range of concentrations (from 2 M to 300 mM). AChE is activated at low substrate concentrations and inhibited at high substrate concentrations. Several rival kinetic models have been developed to try to describe and explain this behavior. One of these models assumes that activation at low substrate concentrations partly results from an acceleration of deacetylation of the acetylated enzyme. To test this hypothesis, we used a monomethylcarbamoylated enzyme, which is considered equivalent to the acylated form of the enzyme and a non-hydrolyzable substrate analog, 4-oxo-N,N,N-trimethylpentanaminium iodide. It appears that this substrate analog increases the decarbamoylation rate by a factor of 2.

Terpenes: Natural Products for Controlling Insects of Importance to Human Health—A Structure-Activity Relationship Study

“…Nevertheless, other studies have hypothesized that aphids, thrips, and probably some insects might have both AChE and BChE [95,105]. In relation to this, an analysis of substrate hydrolysis at different concentrations revealed that insect cholinesterase displays activation at low substrate concentrations similar to that of the vertebrate BChE but demonstrated inhibition at high substrate concentrations similar to the vertebrate AChE [89]. Moreover, Keane and Ryan [96] reported that the inhibitory activity of monoterpenes against electric eel AChE is similar to the effect of inhibiting insect AChE.…”

“…Thus, the control of the activity of this enzyme is very important in determining the insecticidal capacity of natural or synthetic chemicals, with AChE consequently being the primary target of organophosphorus pesticides and carbamates [85,86] as well as of some essential oils and their components [38,49,87,88]. However, the AChE in resistant insects is able to change its active site or create new forms of the enzyme, in response to selective pressure after several years of the application of synthetic pesticides [86,[89][90][91][92][93][94][95][96][97][98].…”

Hydrolysis of pyrethroids by carboxylesterases from Lucilia cuprina and Drosophila melanogaster with active sites modified by in vitro mutagenesis

“…These residues in both insect AChE and vertebrate AChE are thought to operate through non-polar and pelectron interactions with the leaving group of substrates, and when binding to inhibitors (Harel et al, 2000;Brochier et al, 2001). They have been mutagenised in vitro in the AChE of various species to study their effect on interaction with various substrates and inhibitors (http://bioweb.ensam.inra.fr/ESTHER/general?what=mutalign).…”

Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina

“…Structural and mutational studies have shown that the anionic site of cholinesterases functions mainly in the first, enzyme acylation stage of the reaction (Shafferman et al, 1992;Ordentlich et al, 1995Ordentlich et al, , 1996Koellner et al, 2000;Harel et al, 2000;Brochier et al, 2001), particularly in the formation of the non-covalent transition state (Nair et al, 1994). Therefore, mutations of the key residues affect mainly K m rather than k cat .…”