Understanding Allosteric Arginine Mutations Using Macromolecular Rate Theory
Permanent link to Research Commons versionhttps://hdl.handle.net/10289/16359
Enzyme catalysed reaction rates have been traditionally modelled with the Arrhenius and Eyring-Polanyi equations. These models assume that the reaction rate is exponential with temperature, and thus the natural log of the reaction rate versus 1/T is linear. Significant deviations from these models at high temperatures has traditionally been attributed to thermal denaturation. An increasing body of evidence has shown that denaturation alone is insufficient to account for these deviations from Arrhenius behaviour. Macromolecular rate theory (MMRT) accounts for these deviations with the introduction of the activation heat capacity (Δ𝐶𝑃‡) to the rate equation. The activation heat capacity is a consequence of the restriction in conformational freedom along the reaction coordinate, as an enzyme moves from the enzyme-substrate complex to the transition state complex – thus for an enzymatic reaction the activation heat capacity is negative. A non-zero activation heat capacity imparts temperature dependence to the activation entropy and activation enthalpy, introducing curvature to the rate equation independent of thermal denaturation. The activation heat capacity may itself be temperature dependent. MMRT equations of increasing complexity have been developed to reflect this and are suitable for different applications. This thesis explores the effects of allosteric arginine mutations on the temperature dependence of enzyme rates through the lens of MMRT and evolution using the model enzyme MalL. An in-depth analysis of a previously characterised arginine mutant is described along with four additional arginine mutants. The arginine mutants were designed to mimic urea ligand binding across the enzyme surface. These mutants were characterised kinetically and with biophysical methods. Two were further characterised structurally, with high resolution structures being produced. These mutant enzymes showed significant rate improvements at low temperatures, suggesting two possible mechanisms for evolution towards psychrophily. These arginine mutants showed significant improvement in crystallographic resolution, indicating surface arginine mutations may be a general route for crystallographic improvement.
The University of Waikato
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