Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition

The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 °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 ΔH = +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 Km = 3.3 mM, catalytic constant kcat = 1.7 × 103 s−1, bimolecular rate constant ks = 5.1 × 105 M−1 s−1. The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant Ki = 9.1 mM. 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.