Applications of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions

The application of isothermal titration calorimetry (ITC) was tested towards measurements of enzyme kinetics in complex solutions containing high concentrations of proteins. Such investigations are important, due to the increasing interest in biochemical reactions in physiological relevant media as well as the application of enzymes in industrial processes. In contrast to spectral methods, measurements performed with ITC, are independent of the optical properties of solutions, making it possible to measure enzyme kinetics in concentrated solutions of macromolecules. In this study the kinetic properties of hexokinase was investigated in concentrated protein solutions (BSA). It was found, that the quality of the measured kinetic data was independent of protein concentration in the investigated range (0-250 mg BSA ml⁻¹). All results could be accounted for by Michaelis-Menten's approach and both k_cat and K_M decreased with increasing protein concentrations. The decrease in K_M with increasing protein concentration was ascribed to an increase in the ratio of activity coefficients between the native enzyme and the enzyme-substrate complex. The decrease in k_cat with increasing protein concentrations indicates that crowding by BSA effect the conformational changes/rehydration that accompanies catalysis and/or diffusion of product from the enzyme-product complex. The methodology is discussed together with an analysis of the experimental results.