Michelle E. Peterson

The dependence of enzyme activity on temperature: determination and validation of parameters

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.

The effect of temperature on enzyme activity: new insights and their implications

The two established thermal properties of enzymes are their activation energy and their thermal stability. Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive transition, in addition to an irreversible thermal inactivation. Two new thermal parameters, Teq and ΔHeq, describe the active–inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We review here the Model itself, methods for the determination of Teq and ΔHeq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

Eurythermalism and the temperature dependence of enzyme activity

The “Equilibrium Model” has provided new tools for describing and investigating enzyme thermal adaptation. It has been shown that the effect of temperature on enzyme activity is not only governed by ∆G‡cat and ∆G‡inact but also by two new intrinsic parameters, ∆Heq and Teq, which describe the enthalpy and midpoint, respectively, of a reversible equilibrium between active and inactive (but not denatured) forms of enzyme. Twenty‐one enzymes from organisms with a wide range of growth temperatures were characterized using the Equilibrium Model. Statistical analysis indicates that Teq is a better predictor of growth temperature than enzyme stability (∆G‡inact). As expected from the Equilibrium Model, ∆Heq correlates with catalytic temperature tolerance of enzymes and thus can be declared the first intrinsic and quantitative measure of enzyme eurythermalism. Other findings shed light on the evolution of psychrophilic and thermophilic enzymes. The findings suggest that the description of the Equilibrium Model of the effect of temperature on enzyme activity applies to all enzymes regardless of their temperature origins and that its associated parameters, ∆Heq and Teq, are intrinsic and necessary parameters for characterizing the thermal properties of enzymes and their temperature adaptation and evolution.—Lee C. K., Daniel, R. M., Shepherd, C., Saul, D., Cary, S. C., Danson, M. J., Eisenthal, R., Peterson M. E. Eurythermal‐ism and the temperature dependence of enzyme activity FASEB J. 21, 1934–1941 (2007)

New parameters controlling the effect of temperature on enzyme activity

Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive (but not denatured) transition, revealing an additional and reversible mechanism for enzyme activity loss in addition to irreversible thermal inactivation at high temperatures. Two new thermal parameters, T(eq) and DeltaH(eq), describe the active-inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We describe here the Model and its fit to experimental data, methods for the determination of the Equilibrium Model parameters, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

Cryosolvents useful for protein and enzyme studies below −100°C

Abstract For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxyethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase-changes down to at least −160°C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to −125°C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.