Characterization of slow-binding inhibition by isothermal titration calorimetry: The case of urease, a nickel-dependent enzyme

Isothermal titration calorimetry (ITC) can provide a kinetic characterization of an enzyme-catalyzed reaction and inhibition by measuring the heat released or absorbed during the reaction under study. In addition to the classical Michaelis–Menten approach, from which the enzyme kinetic parameters kcat and KM, as well as the inhibition constant KI for a fast-binding inhibitor, can be determined, ITC can be exploited to characterize slow-binding inhibition processes, which occur more slowly than enzyme catalysis and show a time-dependent behavior. In this chapter, two examples are presented describing the characterization of the reversible slow-binding inhibition of urease, a Ni(II)-dependent enzyme involved in human health and agro-environmental issues, using ITC. These examples can help the reader to broaden the application field of ITC, highlighting how it can be used for characterizing virtually any kind of enzymatic inhibition (fast- or slow-binding, reversible or irreversible), a key step in drug discovery and development processes.