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Title: Insight Into Enzyme Catalysis Through The Study of B-Phosphoglucomutase And Uracil DNA Glycosylase
Author: Pollard, Sarah
ISNI:       0000 0001 3493 8994
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2007
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Human uracil DNA glycosylase (UDG) excises uracil bases resulting from cytosine deamination or dUMP incorporation into DNA at a rate of 500 per cell per day. UDG is also important in the study of class-switching in immunoglobulins, and in the development of treatments for tuberculosis, cancer and HIV. This thesis studies tautomerisation of the transition state analogue deoxypseudouridine, which enables determination of strain energy of the reaction. This energy is predicted to be 30-40kJ, which is the biggest strain found in any enzyme to date. The crystal structure and solution conformation of deoxypseudouridine were also solved and deoxypseudouridine was found to be unstrained in the absence ofUDG. His 67 is conserved in the active site ofE. coli UNG (UDG), but has not been studied as part of the enzyme mechanism previously. The pKa of His 67 was determined to find out ifit is cationic, and part of an electrostatic substrate autocatalysis mechanism disrupting the negative charge on Asp 64, and binding the dT4 phosphate in the oligonucleotide substrate. His 67 was found to have a pKa of 6.0-6.5 and is not anticatalytic as suggested in previous QM/MM calculations on human UDG. p-PGM is an example of a phosphate transfer enzyme, crucial for basic processes in the cell such as energy metabolism, signal transduction, and cell division. lIe 110, not in the active site, was mutated to alanine to impair structural tightening in p-PGM. The mutant was used to test the hypothesis that a decrease in structural tightening in enzymes leads to a decrease in kcat • Structural tightening was compared using the transition state analogues G6P:MgF3- and G6P:AIF4-. Greater structural tightening was found in G6P:MgF3- which mimics the geometry and charge of the neartransition state complex in phosphate transfer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available