title: Hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions.
abstract: Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, ΔH) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery. In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (ΔHint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.
authors.affiliation: Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna.
authors.forename: Luca
authors.initials: L
authors.lastname: Mazzei
authors.affiliation: Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
authors.affiliation: Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna; barbara.zambelli@unibo.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Zambelli
mesh_terms: D002151:Calorimetry / Q000379:methods*; D002384:Catalysis; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D014508:Urea / Q000737:chemistry / Q000378:metabolism; D014510:Urease / Q000737:chemistry / Q000378:metabolism
chemical_list: D004798:Enzymes; D014508:Urea; D014510:Urease
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