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J. Biol. Chem., Vol. 276, Issue 21, 18296-18302, May 25, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/21/18296    most recent M005555200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brochier, L. Articles by Fournier, D. Search for Related Content PubMed PubMed Citation Articles by Brochier, L. Articles by Fournier, D. Social Bookmarking                      What's this?

Involvement of Deacylation in Activation of Substrate Hydrolysis by Drosophila Acetylcholinesterase^*

Laure Brochier, Yannick Pontié, Michèle Willson, Sandino Estrada-Mondaca^§, Jerzy Czaplicki^¶, Alain Klaébé, and Didier Fournier

From the  Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologique UMR 5068, Université Paul Sabatier, 31062 Toulouse, France and the ^¶ Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 route de Narbonne, 31077 Toulouse, France

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 µM 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 2.2, suggesting that the substrate molecule bound at the activation site (K_d = 130 ± 47 µM) accelerates deacetylation. These two kinetic parameters are consistent with our analysis of the hydrolysis of the substrate. The location of the active site was investigated by in vitro mutagenesis. We found that this site is located at the rim of the active site gorge. Thus, substrate positioning at the rim of the gorge slows down the entrance of another substrate molecule into the active site gorge (Marcel, V., Estrada-Mondaca, S., Magné, F., Stojan, J., Klaébé, A., and Fournier, D. (2000) J. Biol. Chem. 275, 11603-11609) and also increases the deacylation step. This results in an acceleration of enzyme turnover.

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