Golicnik_2002_Arch.Biochem.Biophys_398_23

The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase EC 3.1.1.8 was investigated at 25 and 37 C The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis The model which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase Stojan et al 1998 FEBS Lett 440 85?88 is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena apparent activation at low substrate concentrations and substrate inhibition by excess of substrate For the analysis of the data in the presence of ethopropazine at two temperatures we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site but the competition at the acylation site was not excluded The proposed reaction scheme revealed upon analysis competitive effects of ethopropazine at both sites at 25 C three enzyme inhibitor dissociation constants could be evaluated at 37 C only two constants could be evaluated Although the model considers both cooperative phenomena it appears that decreased enzyme sensitivity at higher temperature predominantly for the ligands at the peripheral binding site makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible The same reason might also account for one of the paradoxes in cholinesterases activities at 25 C at low substrate concentrations are higher than at 37 C Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328 W82 D70 and Y332 amino acid residues stabilize binding of the inhibitor 2002 Elsevier Science