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Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200-400 g/L of macromolecular crowders such as proteins, ribosomes and lipids. Thus we expanded the ITC application for solutions mimicking the cellular environment by adding various macromolecular crowders. We investigated how these crowders affect the kinetics of trypsin-catalyzed reactions and determined the Michaelis-Menten parameters for hydrolysis of two trypsin substrates: Nα-benzoyl-l-arginine ethyl ester (BAEE) and Nα-benzoyl-dl-arginine β-naphthylamide (BANA). Since ITC enables investigations of complex and turbid solutions with label-free reagents, it seems a perfect technique for kinetic analyses in crowded solutions. ITC also offers the opportunity to control enzyme-crowder and substrate-crowder interactions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis-Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37°C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis-Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays., (Copyright © 2015 Elsevier Inc. All rights reserved.)