To detect traces of insecticides in the environment using biosensors, we engineered Drosophila acetylcholinesterase (AChE) to increase its sensitivity and its rate of phosphorylation or carbamoylation by organophosphates or carbamates. The mutants made by site-directed mutagenesis were expressed in baculovirus. Different strategies were used to obtain these mutants: (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, (iii) Ala-scan analysis of amino acids lining the active site gorge, (iv) mutagenesis at positions detected as important for sensitivity in the Ala-scan analysis and (v) combination of mutations which independently enhance sensitivity. The results highlighted the difficulty of predicting the effect of mutations; this may be due to the structure of the site, a deep gorge with the active serine at the bottom and to allosteric effects between the top and the bottom of the gorge. Nevertheless, the use of these different strategies allowed us to obtain sensitive enzymes. The greatest improvement was for the sensitivity to dichlorvos for which a mutant was 300-fold more sensitive than the Drosophila wild-type enzyme and 288 000-fold more sensitive than the electric eel enzyme, the enzyme commonly used to detect organophosphate and carbamate.
        
Title: Exploration of the Drosophila acetylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes Marcel V, Estrada-Mondaca S, Magne F, Stojan J, Klaebe A, Fournier D Ref: Journal of Biological Chemistry, 275:11603, 2000 : PubMed
Cholinesterases are activated at low substrate concentration, and this is followed by inhibition as the level of substrate increases. However, one of these two components is sometimes lacking. In Drosophila acetylcholinesterase, the two phases are present, allowing both phenomena to be studied. Several kinetic schemes can explain this complex kinetic behavior. Among them, one model assumes that activation results from the binding of a substrate molecule to a non-productive site affecting the entrance of a substrate molecule into the active site. To test this hypothesis, we looked for an inhibitor competitive for activation and we found Triton X-100. Using organophosphates or carbamates as hemisubstrates, we showed that Triton X-100 inhibits or increases phosphorylation or carbamoylation of the enzyme. In vitro mutagenesis of the residues lining the active site gorge allowed us to locate the Triton X-100 binding site at the rim of the gorge with glutamate 107 playing the major role. These results led to the hypothesis that substrate binding at this site affects the entrance of another substrate molecule into the active site cleft.