Tests of predictions made by the Equilibrium Model for the effect of temperature on enzyme activity
Oudshoorn, M. L. (2008). Tests of predictions made by the Equilibrium Model for the effect of temperature on enzyme activity (Thesis, Master of Applied Psychology (MAppPsy)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/2418
Permanent Research Commons link: https://hdl.handle.net/10289/2418
The Classical Model describing the effects of temperature on enzyme activity consists of two processes: the catalytic reaction defined by ΔG cat and irreversible inactivation defined by ΔG inact, this model however, does not account for the observed temperature- dependant behaviour of enzymes. The recent development of the Equilibrium Model is governed not only by ΔG cat and ΔG inact but also by two new intrinsic parameters ΔHeq and Teq, which describe the enthalpy and the temperature of the midpoint, respectively, of a active and reversibly inactive enzyme transition. Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological and environmental aspects of life to temperature in a way that has not been previously possible. The Equilibrium Model is therefore a more complete and accurate description of the effects of temperature on enzymes, it has provided new tools for describing and investigating enzyme thermal adaptation and possibly new biotechnological tools. The effects of the incorporating in the new Model of the parameters Teq and ΔH eq yield major differences from the Classical Model, with simulated data calculated according to the Equilibrium Model fitting well to experimental data and showing an initial rate temperature optimum that is independent of assay duration. Simulated data simulated according to the Classical Model can not be fitted to experimental data. All enzymes so far studied (gt30) display behaviour predicted by the Equilibrium Model. The research described here has set out to: experimentally test observations made by Eisenthal et al., on the basis of enzyme reactor data simulated according to the Equilibrium Model; to test the Equilibrium Model using an unusual (rapidly renaturable) enzyme, RNAase; and to test the proposed molecular basis of the Equilibrium Model by examining the effect of a change at the enzymes active site. The experimental results gathered here on the effect of time and temperature on enzyme reactor output confirm the predictions made by Eisenthal et al. (2006) and indicate that the Equilibrium Model can be a useful aid in predicting reactor performance. The Equilibrium Model depends upon the acquisition of data on the variation of the Vmax of an enzyme with time and temperature, and the non-ideal behaviour of RNase A made it impossible to collect such data for this enzyme, as a result the Equilibrium Model could not be applied. The disulfide bond within the active site cleft of A.k 1 protease was cleaved as a probe of the mechanism of the Equilibrium Model, which is proposed to arise from molecular changes at the enzymes active site. Support for the proposed mechanism was gained through the comparison of experimentally determined temperature dependence of the native and reduced forms of the enzyme and application of this data to the Equilibrium Model.
The University of Waikato
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