Enzymatic activity assay of d-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Abstract

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of dhydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.