Elucidating the Complexity of Dynamic Networks in Enzyme Catalysis

  • Samuel Winter

Student thesis: Doctoral ThesisPhD


Since the 20th century, there have been big advancements in our understanding of enzymes, more recently there has been a divergence in ideas surrounding how their catalysis is influenced by temperature. At a rudimentary understanding of an enzyme’s relationship to temperature, we acknowledge that rate of turnover increases with temperature until an optimum temperature is reached (Topt) and at temperatures past Topt this rate will decrease. Our initial understanding is that the drop in rate is due to enzyme denaturation, which we still believe to be the main determining factor in the majority of cases. However, more and more research has shown that denaturation alone is sometimes not enough to explain the phenomena.
There has been a recent boom in enzymatic research to try and identify the reasoning for the unique temperature dependence of the rate of enzyme turnover. One idea that is explored in this thesis, is that the change in Gibbs free energy from ground state to transition state (ΔG‡) is temperature dependent itself. When we make this assertion, we introduce the term (ΔCp‡) which is the change in heat capacity from ground state to transition state. The aforementioned idea is known as macromolecular rate theory (MMRT) and the changes in the key ΔCp‡ term in response to different factors has been studied at great length by several different research groups since 2013 and there is mounting evidence to suggest that ΔCp‡ could be the determinant factor in the curvature of temperature dependant enzyme catalysis. The biggest contribution to the ΔCp‡ term is notionally altered vibrational motion. In this thesis we explored how the temperature dependence of catalysis, in a promiscuous thermophilic enzyme, sulfolobus solfataricus glucose dehydrogenase (ssGDH), changes in response to different substrates. We studied the change in ΔCp‡ using experimental techniques and we found that there were significant differences in the temperature dependence of catalysis when different substrates were bound. We analysed these differences using red edge excitation shift (REES) experiments and molecular dynamics simulations and found that these changes were accompanied by dynamical changes in the enzyme. We also generated crystal structures of ssGDH, which we used for generating the 3D structure of the enzyme using X-ray crystallography. We also investigated internal protein motions in a medically relevant enzyme, SARS COV-2 main protease (Mpro). We investigated how motions of amino acids change in MPro in the presence of different inhibitors as a method of understanding how motions change in response to inhibitors. This is with the ultimate aim of being able to better design inhibitors and simultaneously understand the role of motion within enzymes. The work in this thesis is aimed to better understand enzymes and help future studies for tuning an enzyme’s temperature dependencies and also in better understanding inhibitor-enzyme interactions.
Date of Award12 Oct 2022
Original languageEnglish
Awarding Institution
  • University of Bath
SponsorsESF and EPSRC
SupervisorJean Van Den Elsen (Supervisor) & Susan Crennell (Supervisor)

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