Precise Evaluation of Enzyme Activity using Isothermal Titration Calorimetry

Abstract

Enzymes, the catalysts of biological systems, are remarkable molecular devices that determine the patterns of chemical transformations. For many decades, a great deal of pure and applied studies have been devoted to enzyme reactions, as the mechanisms underlying the special characteristics of the reactions, such as high specificity and high efficiency, have not only attracted scientific interest but also indicated the possibility of employing enzymes as highly useful catalysts in many application fields. Quantitative evaluation of the catalytic activity of enzymes is inevitably important to reveal the enzymes' reaction mechanisms and to use the catalysts most effectively. To evaluate enzyme kinetics, it is necessary that the transformation of a substrate into a product is accompanied with an observable event. Since the change in enthalpy is one of a reaction's general features, several approaches have been employed to monitor enzyme-catalyzed reactions using calorimetry.1-17) The heat generated as the reaction proceeds is a direct and sensitive observable quantity. Because it allows the direct determination of the reaction rate, which indicates the enzyme activity itself, calorimetry is expected to provide a general and effective way to evaluate enzyme activity. ITC provides one possibility for detecting the catalytic reaction heat as a function of time with high sensitivity and reproducibility. Two calorimetric variables, the compensation power and its integral, can be determined directly and precisely by this method.13-15) Under hydrolytic conditions, the combination of a calorimetric LineweaverBurk plot with these two variables and the non-linear least-squares method was found to be effective for determining enzymatic parameters precisely.13-15) The kinetic parameters, kcat and KM, obtained from calorimetric observables, clearly indicated that the enzyme-catalyzed hydrolysis reaction is well approximated by a simple Michaelis-Menten equation.13-15) Tradit ionally, ITC analysis has used data obtained only after the enzyme solution has been fully titrated.13, 14) However, since the enzyme reaction occurs in the cell even during titration, and since the precise total enzyme concentration in the cell is determined by the titration program, it is possible to use the experimental data gathered during titration for the analysis. To determine the reaction heat and enzyme parameters more precisely, the traditional method has been modified to treat all of the hydrolysis data observed by ITC.15) In addition to activity measurement, inhibition studies of different enzyme-catalyzed hydrolysis reactions have used ITC with great success.8-11,14,15) In most models of