Abstract
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.
Highlights
Cholinesterases belong to the family of serine hydrolases
E is the free enzyme, EA the acylated enzyme, and SE represents the binding of a substrate molecule onto the activation site. ka represents the bimolecular rate constant for acylation and kcat the rate constant for deacylation. a and b coefficients represent the effect of the activation site occupation by a substrate molecule on deacylation and acylation respectively
In vitro expression of a soluble cholinesterase, devoid of any lipid anchor, allowed kinetic studies to be performed in the absence of detergent, activation to be demonstrated at low substrate concentration [9], and the effect of Triton X-100 to be studied
Summary
Chemicals and Source of Enzyme—Triton X-100, polyethylene glycol, reduced Triton X-100, edrophonium, and propidium were from Sigma. As Triton X-100 is a mixture of two chain lengths (n ϭ 9 and 10), its concentration will be expressed in g1⁄7literϪ1. Hydrolysis of acetylthiocholine iodide was studied spectrophotometrically at 412 nm using the method of Ellman et al [20], at substrate concentrations from 2 M to 200 mM in 1-cm path length cuvettes using a Safas DES spectrophotometer. Kobs is the number of micromoles of product.
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