Enzymatic activity assay of d-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of d-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/ti...

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Bibliographic Details
Published in:Journal of biochemical and biophysical methods 2006-04, Vol.67 (1), p.57-66
Main Authors: Andújar-Sánchez, Montserrat, Las Heras-Vázquez, Francisco Javier, Clemente-Jiménez, Josefa María, Martínez-Rodríguez, Sergio, Camara-Artigas, Ana, Rodríguez-Vico, Felipe, Jara-Pérez, Vicente
Format: Article
Language:English
Subjects:
Activation parameters
Activity assay
Amidohydrolases - chemistry
Calorimetry
d-hydantoinase
Hydrolysis
Isothermal titration calorimetry
Kinetics
Recombinant Proteins - chemistry
Rhizobium - enzymology
Substrate Specificity
Thermodynamics
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Summary:Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of d-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant ( k cat), Michaelis constant ( K M), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.
ISSN:0165-022X
1872-857X
DOI:10.1016/j.jbbm.2006.01.002