Measuring heat to understanding catalysis: isothermal titration

Isothermal titration calorimetry (ITC) is a widely used technique to characterize the thermodynamics of biochemical reactions, as it uses the heat released or absorbed upon virtually all reactions as an internal probe. Indeed, the measured heat is related to the amount of reacting molecules and to the rate of the reaction of interest. The ability of this technique to derive the thermodynamic parameters of the biochemical processes makes it an invaluable and powerful technique to study biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, ΔH) of enzymatic reactions. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. These properties render ITC a unique tool to study enzyme kinetics in applications such as drug discovery. These features will be described and discussed together with some applications to specific cases of enzyme catalysis and inhibition. A general guideline to choose the right procedure according to the system under analysis is given, together with some experimental tips on how to adjust the conditions for increased data quality. The method to analyze the obtained raw ITC curves and to derive the kinetic parameters and inhibition constants is described.

10/05/2024 Prof. Barbara Zambelli Dipartimento di Farmacia e Biotecnologie Università di Bologna

© Università degli Studi di Roma "La Sapienza" - Piazzale Aldo Moro 5, 00185 Roma