Abstract
Isothermal titration calorimetry (ITC) involves accurately measuring the heat that is released or absorbed in real time when one solution is titrated into another. This technique is usually used to measure the thermodynamics of binding reactions. However, there is mounting interest in using it to measure reaction kinetics, particularly enzymatic catalysis. This application of ITC has been steadily growing for the past two decades, and the method is proving to be sensitive, generally applicable, and capable of providing information on enzyme activity that is difficult to obtain using traditional biochemical assays. This review aims to give a broad overview of the use of ITC to measure enzyme kinetics. It describes several different classes of ITC experiment, their strengths and weaknesses, and recent methodological advancements. A summary of applications in the literature is given and several examples where ITC has been used to investigate challenging aspects of enzyme behavior are presented in more detail. These include examples of allostery, where small-molecule binding outside the active site modulates activity. We describe the use of ITC to measure the strength, mode (i.e., competitive, uncompetitive, or mixed), and association and dissociation kinetics of enzyme inhibitors. Further, we provide examples of ITC applied to complex, heterogeneous mixtures, such as insoluble substrates and live cells. These studies exemplify the wide range of problems where ITC can provide answers, and illustrate the versatility of the technique and potential for future development and applications.