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.

Temperature and the catalytic activity of enzymes: A fresh understanding

“…This is known from the experimental observation that enzyme initial rates do in fact decline at high temperatures (see Fig. 6 for an example), and at all temperatures the E act /E inact equilibration is faster than the time needed to start the reaction in a stirred spectrophotometer cuvette (of the order of 1-3 s), and the line of product vs time extrapolates back to zero [7][8][9][10]. So far, all enzymes we have studied that obey Michaelis-Menten kinetics follow this Model [7][8][9], irrespective of mechanism or structure (e.g., Table 1), and all have a temperature optimum for initial rates, as expected from the Model.…”

Photosynthesis and lipid composition of the Antarctic endemic rhodophyte Palmaria decipiens: effects of changing light and temperature levels

“…In general, low temperatures slow down enzymatic reactions and synthetic pathways and result in a decrease in membrane Xuidity (Gurr et al 2002;Peterson et al 2007). To photosynthesizing organisms, as e.g.…”

Enzymic Approach to Eurythermalism of Alvinella pompejana and Its Episymbionts

“…The recently proposed and experimentally validated equilibrium model (13,27,28) has provided a solution to the above problem. By incorporating a reversibly inactive form of enzyme (E inact ) in addition to the active form (E act ) and the irreversibly denatured form (X), the equilibrium model proposes that an equilibrium exists between the active and inactive forms of enzyme, as expressed in the following equation:…”

“…In general terms, enzyme assays were carried out as described previously (27). All of the enzyme assays performed in this work were continuous spectrophotometric assays measured with a Thermo Spectronic Helios Gamma spectrophotometer equipped with a Thermo Spectronic single-cell Peltier effect cuvette holder.…”