The molecular basis of the effect of temperature on enzyme activity.

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

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 deltaG(double dagger)(cat) and deltaG(double dagger)(inact) but also by two new intrinsic parameters, deltaH(eq) and T(eq), 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 T(eq) is a better predictor of growth temperature than enzyme stability (deltaG(double dagger)(inact)). As expected from the Equilibrium Model, deltaH(eq) 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, deltaH(eq) and T(eq), are intrinsic and necessary parameters for characterizing the thermal properties of enzymes and their temperature adaptation and evolution.

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.

Rapid increases in soil pH solubilise organic matter, dramatically increase denitrification potential and strongly stimulate microorganisms from the Firmicutes phylum.

Rapid and transient changes in pH frequently occur in soil, impacting dissolved organic matter (DOM) and other chemical attributes such as redox and oxygen conditions. Although we have detailed knowledge on microbial adaptation to long-term pH changes, little is known about the response of soil microbial communities to rapid pH change, nor how excess DOM might affect key aspects of microbial N processing. We used potassium hydroxide (KOH) to induce a range of soil pH changes likely to be observed after livestock urine or urea fertilizer application to soil. We also focus on nitrate reductive processes by incubating microcosms under anaerobic conditions for up to 48 h. Soil pH was elevated from 4.7 to 6.7, 8.3 or 8.8, and up to 240-fold higher DOM was mobilized by KOH compared to the controls. This increased microbial metabolism but there was no correlation between DOM concentrations and CO2 respiration nor N-metabolism rates. Microbial communities became dominated by Firmicutes bacteria within 16 h, while few changes were observed in the fungal communities. Changes in N-biogeochemistry were rapid and denitrification enzyme activity (DEA) increased up to 25-fold with the highest rates occurring in microcosms at pH 8.3 that had been incubated for 24-hour prior to measuring DEA. Nitrous oxide reductase was inactive in the pH 4.7 controls but at pH 8.3 the reduction rates exceeded 3,000 ng N2-N g-1 h-1 in the presence of native DOM. Evidence for dissimilatory nitrate reduction to ammonium and/or organic matter mineralisation was observed with ammonium increasing to concentrations up to 10 times the original native soil concentrations while significant concentrations of nitrate were utilised. Pure isolates from the microcosms were dominated by Bacillus spp. and exhibited varying nitrate reductive potential.

The thermal behaviour of enzyme activity: implications for biotechnology.

The way that enzymes respond to temperature is fundamental to many areas of biotechnology. This has long been explained in terms of enzyme stability and catalytic activation energy, but recent observations of enzyme behaviour suggest that this picture is incomplete. We have developed and experimentally validated a new model to describe the effect of temperature on enzymes; this model incorporates additional fundamental parameters that enable a complete description of the effects of temperature on enzyme activity. In this article, we consider the biotechnological implications of this model in the areas of enzyme engineering, enzyme reactor operation and the selection and/or screening of useful enzymes from the environment.

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 Delta Heq, 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 Delta Heq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

A new intrinsic thermal parameter for enzymes reveals true temperature optima.

Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, T(eq), arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source ("evolved") temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, T(eq) is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on T(eq) with respect to enzyme temperature/activity effects to be more important than on thermal stability. T(eq) is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution.