Understanding allosteric enzyme regulation using macromolecular rate theory
Hamill, C. J. (2020). Understanding allosteric enzyme regulation using macromolecular rate theory (Thesis, Master of Science (Research) (MSc(Research))). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/13541
Permanent Research Commons link: https://hdl.handle.net/10289/13541
Enzyme catalysed reaction rates produce a curved temperature dependence with a temperature optimum, Topt. Traditionally enzyme temperature dependence has been modelled by the Arrhenius and Eyring-Polanyi equations. Deviations from these models at elevated temperatures have traditionally been attributed to enzyme denaturation. Increasing evidence shows that enzyme denaturation is insufficient to fully explain the curvature for enzyme catalysed rates with temperature. Macromolecular rate theory (MMRT) accounts for these deviations from early models by introducing the concept of heat capacity (Cp). During an enzymatic reaction, the heat capacity is reduced as the transition state complex forms (due to enzyme rigidification), resulting in an overall negative change in heat capacity (ΔCp^‡) for the reaction. Heat capacity is a function of the number and energies of the vibrational modes for the individual molecules. Enzyme-ligand binding will influence the energies of the vibrational modes of the molecular complex therefore affecting an enzymes’ heat capacity and its catalytic rate via ΔCp^‡. Notably, a form of enzyme regulation (called allostery) utilises ligand binding at a site other than the active site to effect changes in catalytic rate. The underlying mechanisms of allostery are poorly understood. Here, the mechanisms of allostery are explored using a model isomaltase enzyme and MMRT to investigate changes in heat capacity. Several mutants of the model MalL enzyme were designed, produced and purified. These mutations aimed to either alter the structural integrity or mimic ligand binding in the C-terminal domain. Using a Stopped-Flow apparatus, these mutants were characterised kinetically and using crystallographic techniques, two structures were solved at high resolution. Targeted mutations approximately 30 Å from the active site showed significant changes in enzyme rate. Overall, the two new structures of MalL were very similar to the apo-enzyme showing only very minor structural deviations. Changes to the rate can thus be attributed to changes in enzyme dynamics and heat capacity. These results are a promising first step in linking changes in enzymatic activity with changes to enzyme dynamics and heat capacity, using MMRT as a model.
The University of Waikato
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