title: Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.
abstract: The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.
authors.affiliation: Centre de Recherches du Service de Santé des Armées, Unité d'Enzymologie, La Tronche, France.
authors.forename: A
authors.initials: A
authors.lastname: Weingand-Ziadé
authors.forename: F
authors.initials: F
authors.lastname: Renault
authors.forename: P
authors.initials: P
authors.lastname: Masson
mesh_terms: D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000235:genetics; D002092:Butyrylthiocholine / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011312:Pressure; D011487:Protein Conformation; D013696:Temperature
chemical_list: D002092:Butyrylthiocholine; D002091:Butyrylcholinesterase