7th International Conference on Protein Stabilization 2007 7th International Conference on Protein Stabilization 2007 Independent Meeting held at the University of Exeter, Exeter, U.K., 11–14 April 2007. Organized and Edited by J. Littlechild (Exeter, U.K.). New parameters controlling the effect of temperature on enzyme activity R.M. Daniel* 1 , M.J. Danson†, R. Eisenthal‡, C.K. Lee* and M.E. Peterson* *Thermophile Research Unit, School of Science and Engineering, University of Waikato, Hamilton, New Zealand, †Centre for Extremophile Research and Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K., and ‡Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K. Abstract 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 H 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. Introduction Chemical reactions go faster as the temperature rises, but the inactivation of enzymes through protein denaturation also increases with temperature [1,2]. Until recently, the influence of temperature on enzyme-catalysed reactions has been understood in terms of two parameters, G ‡ cat and G ‡ inact , which govern the temperature-dependence of the catalytic rate and the rate of enzyme denaturation respectively. Since denaturation is relatively slow, at very short assay durations (where denaturation is negligible), this ‘Classical Model’ has no optimum; at longer durations, the temperature optimum is dependent on assay duration. By inserting plausible values for G ‡ cat and G ‡ inact , it is possible to plot the effect of time and temperature on enzyme activity (Figure 1A). Any discrepancies between experimental data and this model have been difficult to quantify because activity and stability are usually measured under different conditions. The Equilibrium Model Recent observations indicate that the actual temperature- dependent behaviour of enzymes cannot be fully described Key words: biotechnology, enzyme activity, enzyme inactivation, Equilibrium Model, stability, temperature optimum. 1 To whom correspondence should be addressed (email r.daniel@waikato.ac.nz). in terms of G ‡ cat and G ‡ inact ; in particular, enzyme activity reductions at high temperatures are greater than predicted from their stability [3,4]. We have therefore developed a theoretical model [5] that incorporates an intermediate reversible equilibrium (eqn 1) between an inactive (but not denatured) form of the enzyme and the active form. X denotes the irreversibly denatured form of the enzyme. E act Keq  E inact kinact −−−→ X (1) The equilibrium is characterized in terms of the enthalpy of the equilibrium, H eq , and a new thermal parameter, T eq , which is the temperature at which the concentrations of the active enzyme (E act ) and the inactive enzyme (E inact ) are equal and thus is the thermal equivalent of K m . ln( K eq ) = H eq R  1 T eq − 1 T  (2) When plausible values for H eq and T eq , together with the same values for G ‡ cat and G ‡ inact as used for the Classical Model, are incorporated into simulated progress curves, it be- comes apparent that, unlike the Classical Model (Figure 1A), the Equilibrium Model typically gives rise to a ‘tent-shaped’ three-dimensional rate–temperature–time profile (Figure 1B) C  The Authors Journal compilation C  2007 Biochemical Society 1543 Biochemical Society Transactions www.biochemsoctrans.org