Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases

Isothermal titration calorimetry has been applied to the determination of t he kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat re leased during the hydrolysis of chitin glycosidic bonds. Experiments were c arried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Differen t experimental temperatures were used in order to compare the thermodepende nce of the activity of two chitinases from the psychrophile Arthrobacter sp . TAD20 and of chitinase A from the mesophile Serratia marcescens. The meth od allowed to determine unequivocally the catalytic rate constant k(cat), t he activation energy (E-a) and the thermodynamic activation parameters (Del taG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble s ubstrate. The catalytic activity has also been determined on insoluble chit in, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chit inases was lower than that observed with the mesophilic counterpart. (C) 20 01 Elsevier Science B.V. All rights reserved.