Metal fluorides (MgF3-, AIF4 - and BeF3-) have been used to mimic the transition state and ground state of phosphate transfer enzymes. The aim of this thesis is to study how phosphate transfer enzymes are able to achieve rate enhancements of 1021 over the uncatalysed reaction, using the full complement of metal fluoride analogues. NMR and X-ray crystallography have been used synchronically to extend the current knowledge of phosphate transfer in β-phosphoglucomutase (βPGM) and phosphoserine phosphatase (PSP). The charge balance theory proposes that the active site of phosphate transfer enzymes is set-up to neutralise the negative charge on the substrate and has been proposed in the literature using phosphoglycerate kinase. Chapter 3 of this thesis shows that the charge balance hypothesis holds true for ~PGM and highlights the importance of local charge in the active site. Chapters 4 and 5 establish the use of BeF3- as a ground state analogue in ~PGM. Complexes in the absence and presence of substrate, have been fully characterised by 19F NMR, IH and 15N chemical shift analysis including backbone assignment and X-ray crystallography. Key residues involved in cap and core domain closure have been recognised through this analysis. A minor conformer of the βPGM:MgF3-:G6P TSA complex was observed in the 19F NMR spectrum. Chapter 6 establishes the nature of this complex as arising from a change in geometry from trigonal bipyramidal to octahedral geometry around the central magnesium ion in the metal fluoride moiety. The full complement of metal fluoride complexes of PSP have been formed and characterised by 19F NMR in chapter 7. The 19F chemical shift and sum isotope shift for each fluoride has been recorded in each complex and has been compared to the available X-ray crystal structures to establish that the NMR and crystallography data report on the same structures.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:574570 |
| Date | January 2011 |
| Creators | Griffin, Joanna Louise |
| Publisher | University of Sheffield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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