AbstractThe concept of the heat capacity of an enzyme changing over its reaction coordinate (∆Cp‡) had not been considered before 2013. However, the large size and associated dynamics of macromolecules such as enzymes, lends them to a large heat capacity, a property that could change significantly during enzyme turnover. The macromolecular rate theory (MMRT)1 proposed that the temperature-dependence of changes in enthalpy and entropy associated with enzyme turnover, occurring as a result of a change in heat capacity, can lead to curvature in the temperature-dependence of enzymatic rates. Notionally, the main contribution to ∆Cp‡ is altered vibrational motion. The work presented in this thesis aimed to validate the MMRT, and to investigate the utilization of ∆Cp‡ as a probe of enzyme dynamics linked to catalysis. This was achieved by perturbing the vibrational motion of two different enzyme systems, using high-pressure, viscosity and kinetic isotope effects, and monitoring the subsequent effect of this on the value of ∆Cp‡. The results demonstrate that the value of ∆Cp‡ is sensitive to perturbations in enzyme dynamics, simultaneously validating the MMRT, and the importance of dynamical contributions to catalysis. The approach is also applied to a medically relevant enzyme, with values of ∆Cp‡ revealing that the catalysis of this system is sensitive to its membrane environment. This directed further analysis of the influence of the membrane environment on its turnover. Consequently, molecular dynamic simulations were used to identify previously unknown substrate entrances, and thus new drug targets, for the enzyme. The necessity to acknowledge the effect of ∆Cp‡ when investigating the temperature-dependence of enzymatic rates, is therefore highlighted by this work. Ultimately, it will aid in future work to engineer the optimum temperature, and rate at the optimum temperature, of enzymes. This will be valuable in both medical and industrial enzyme applications.
|Date of Award||2019|
|Supervisor||David Leak (Supervisor) & Christopher Pudney (Supervisor)|
A Biophysical Approach to Understanding the Temperature-dependence of Enzyme Catalysis
Jones, H. (Author). 2019
Student thesis: Doctoral Thesis › PhD