Exploration of the Drosophila acetylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes

Cholinesterases are activated at low substrate concentration, and this is f ollowed by inhibition as the level of substrate increases. However, one of these two components is sometimes lacking. In Drosophila acetylcholinestera se, the two phases are present, allowing both phenomena to be studied. Seve ral kinetic schemes can explain this complex kinetic behavior. Among them, one model assumes that activation results from the binding of a substrate m olecule to a non-productive site affecting the entrance of a substrate mole cule into the active site. To test this hypothesis, we looked for an inhibi tor competitive for activation and we found Triton X-100. Using organophosp hates or carbamates as hemisubstrates, we showed that Triton X-100 inhibits or increases phosphorylation or carbamoylation of the enzyme. In vitro mut agenesis 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 pl aying the major role. These results led to the hypothesis that substrate bi nding at this site affects the entrance of another substrate molecule into the active site cleft.