title: Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.
abstract: Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.
authors.affiliation: Laboratory of Biochemistry, Institute of Chemistry, University of Liege, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: Lonhienne
authors.forename: E
authors.initials: E
authors.lastname: Baise
authors.forename: G
authors.initials: G
authors.lastname: Feller
authors.forename: V
authors.initials: V
authors.lastname: Bouriotis
authors.forename: C
authors.initials: C
authors.lastname: Gerday
mesh_terms: D001173:Arthrobacter / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods*; D002236:Carbohydrate Conformation; D002384:Catalysis; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000378:metabolism*; D004798:Enzymes / Q000378:metabolism*; D005615:Freezing; D046911:Macromolecular Substances; D012706:Serratia marcescens / Q000201:enzymology; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes; D046911:Macromolecular Substances; D002686:Chitin; D002688:Chitinases