Functional consequences of changing structure, dynamics, and free-energy landscapes of phosphoenolpyruvate carboxykinases
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The complex relationship between structure, dynamics, and function will afford insights into the operational details of enzymes. To understand, regulate, and create enzymes, this complex nature must be comprehensively understood. In order to do so, perturbing a well-characterized enzyme in order to discern how changes in primary sequence, alter the structure and dynamics will begin to uncover these details. These modifications and their functional consequences will explain not only how the model system behaves, but also can be extrapolated to better understand the foundational principles of enzymes in general. These studies carried out in this thesis utilize a well-characterized enzyme, phosphoenolypruvate carboxykinase (PEPCK), as a structural and functional scaffold. Three studies investigate increasing degrees of change to this scaffold. The first perturbations of this scaffold start with a single point mutant that imparts a hysteretic kinetic behaviour in order to understand how an allosterically localized specific residue can control global dynamics. The second study examines three isozymes of PEPCK from different organisms that have adapted to different thermal niches. This study attempts to understand how full sequence adaptations tune enzyme structure and dynamics to different thermal availability, balancing dynamics with turnover. The final study is a comprehensive evaluation of the third class of PEPCK, a pyrophosphate-dependent enzyme, which is structurally and functionally distinct from the classical ATP and GTP-dependent PEPCKs. This study acts as a starting point for further investigations of this variant of PEPCK while also highlighting how adding different domains/lobes to a common central scaffold can drastically alter function. An Appendix is also included which demonstrates how a series of related inhibitors can have drastic differences in potency for a potential therapeutic target, the enzyme cytosine triphosphate synthase.
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Matthew McLeod (2020). Functional consequences of changing structure, dynamics, and free-energy landscapes of phosphoenolpyruvate carboxykinases. UWSpace. http://hdl.handle.net/10012/16332