Biochemical properties of recombinant acetylcholinesterases with amino acid substitutions in the active site

Several amino acid substitutions causing insensitivity have been found in the active site of Ace-paralogous acetylcholinesterase (AP-AChE); Gly119Ser (Culex pipiens, Anopheles gambiae), Ala201Ser (Aphis gossypii), Phe290Val (Nephotettix cincticeps), Ser331Phe (Myzus persicae, A. gossypii), Phe331Cys (Tetranychus urticae) and Phe331Trp (Cx. tritaeniorhynchus). To confirm the responsibility of these substitutions to the insensitivity, the six substitutions were introduced into the AP-AChE cDNA of Cx. tritaeniorhynchus resistant strain (Toyama), and their biochemical properties were examined by using a baculovirus-insect cell system. The substitution Gly119Ser gave the enzyme a high level of insensitivity to carbamate insecticides but a slight insensitivity to organophosphates. On the other hand, an amino acid substitution Phe331Trp located in the acyl pocket induced a very high level of insensitivity to organophosphates and ten times lower insensitivity to carbamates. The amino acid replacement appear to render the acyl pocket less hydrophobic and smaller, and then alter the accessibility of the substrates and inhibitors to this site.