Flow microcalorimetric study of enzyme reactions

The enzymatic hydrolysis of phenyl acetate, catalysed by arylesterase/paraoxonase (EC 3.1.8.1) was studied at 37 ° C in Tris buffer, pH 8, by spectrophotometry and flow microcalorimetry, using an enzyme purified from human serum. After correction for buffer protonation and product ionization, the hydrolysis reaction was found to be slightly endothermic, with ΔH=8.2 kJ mol−1. Microcalorimetric data were analysed with the integrated Michaelis equation to give the kinetic parameters of the enzyme: Michaelis constant Km=2.4 mM, catalytic constant kcat=2.4×103 s−1, bimolecular rate constant ks=1.0×106 M−1 s−1. These results were in agreement with the spectrophotometric method. This study confirms the usefulness of microcalorimetry in the field of enzyme kinetics.