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J Biol Chem, Vol. 275, Issue 16, 11603-11609, April 21, 2000

Exploration of the Drosophila Acetylcholinesterase Substrate Activation Site Using a Reversible Inhibitor (Triton X-100) and Mutated Enzymes^*

Véronique Marcel, Sandino Estrada-Mondaca^§, Frédéric Magné, Jure Stojan^¶, Alain Klaébé, and Didier Fournier

From the  Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique, ESA 5068, Groupe de Biochimie des Protéines, Université Paul Sabatier, 31062 Toulouse, France and the ^¶ Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia

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.

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