title: The dependence of enzyme activity on temperature: determination and validation of parameters.
abstract: Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.
authors.affiliation: Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Peterson
authors.forename: Roy M
authors.initials: RM
authors.lastname: Daniel
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Danson
authors.forename: Robert
authors.initials: R
authors.lastname: Eisenthal
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism*; D000626:Aminopeptidases / Q000378:metabolism*; D008954:Models, Biological; D011489:Protein Denaturation; D013696:Temperature; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000135:Acid Phosphatase; D000626:Aminopeptidases; D001618:beta-Lactamases