title: Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition.
abstract: The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent results: Michaelis constant K(m)=3.3mM, catalytic constant k(cat)=1.7 x 10(3)s(-1), bimolecular rate constant k(s)=5.1 x 10(5)M(-1)s(-1). The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant K(i)=9.1mM. Betaine had a slightly higher affinity for the enzyme, but the inhibition was only partial. This study confirms the usefulness of microcalorimetry for the kinetic study of enzymes and their inhibitors.
authors.affiliation: Service de Pharmacologie-Toxicologie, Hôpital Dupuytren, 87042 Limoges, France. jean.debord@unilim.fr
authors.forename: Jean
authors.initials: J
authors.lastname: Debord
authors.forename: Bernard
authors.initials: B
authors.lastname: Verneuil
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Bollinger
authors.forename: Louis
authors.initials: L
authors.lastname: Merle
authors.forename: Thierry
authors.initials: T
authors.lastname: Dantoine
mesh_terms: D000818:Animals; D001622:Betaine / Q000494:pharmacology; D002091:Butyrylcholinesterase / Q000097:blood / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods*; D002794:Choline / Q000494:pharmacology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D006736:Horses; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D013816:Thermodynamics
chemical_list: D002800:Cholinesterase Inhibitors; D001622:Betaine; D002091:Butyrylcholinesterase; D002794:Choline