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Title: Investigations of the dynamics and mechanism of β-phosphoglucomutase
Author: Robertson, Angus
ISNI:       0000 0004 7657 3120
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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This thesis uses a multidisciplinary approach of nuclear magnetic resonance (NMR) spectroscopy, Xray crystallography, and enzyme kinetics to further investigate how β-Phosphoglucomutase (βPGM; EC , an archetypal phosphoryl transfer enzyme from the HAD superfamily, catalyses the inter-conversion of β-glucose 1-phosphate (βG1P) with glucose 6-phosphate (G6P) via a β-glucose 1,6-phosphate (βG16P) intermediate. The use of metal fluorides to mimic positions along the reaction coordinate of phosphoryl transfer enzymes has been well established and allows for a critical investigation of the the role of enzyme dynamics, electrostatics, conformation, and intrinsic organization of the enzyme in catalysis. In a series of papers, this thesis demonstrates several elements of how βPGM has evolved to perform its function. Firstly, mutation of the enzymatic general acid-base (GAB) allowed the investigation of native substrate in the active site of the enzyme. This ground state model was closed around the substrate, with transferring phosphate and nucleophile in van der Waals contact, but without overall transition state architecture. Furthermore, a weakened magnesium affinity in this ground state suggests a mechanism for dissociation of such a high affinity ligand, essential for efficient catalysis. Secondly, using the same GAB mutation, the role of proton transfer in phosphoryl transfer reactions is investigated in pre- and post- proton transfer models. Using a combination of NMR, X-ray crystallography and DFT calculation, it is determined that the proton transfer event is not synchronous with phosphorous transfer, and several key themes are elucidated; before, during, and after the chemical transfer. Each of which contribute to the capacity of βPGM to break and form phosphate monoester bonds on a viable timescale. Thirdly, a mechanism is presented to explain a previously modelled enzymatic lag phase prior to steady state catalysis. Mutation of a key arginine residue is sufficient to alleviate this lag phase and does not perturb the chemical step of the reaction which indicates that such perturbations are not transmitted through substrate to the catalytic center. Finally, it is observed that the phospho-enzyme state of βPGM, when compared to a specific phosphatase (phosphoserine phosphatase (PSP)), displays several features in order to stabilize the phospho-enzyme state that are not present in PSP. Together these features further describe how βPGM has evolved both specificity and to achieve high levels of catalytic rate enhancement.
Supervisor: Waltho, Jonathan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available