Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733561
Title: Molecular mechanism and inhibition of human ATIC
Author: Cox, Heather Louise
ISNI:       0000 0004 6493 7329
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2014
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Abstract:
ATIC is a dimeric bifunctional protein, with two distinct catalytic domains located on each monomer. These catalyse the penultimate and final steps of the de novo purine biosynthetic pathway. It has been extensively studied as a target for chemotherapeutics and anti-viral inhibitors, but the enzyme has not been fully characterized. An ordered binding model for AICAR transformylase has been deduced using a new two-dimensional high-throughput assay; from this the true KM for substrate and cofactor have been measured. The binding affinities for various ligands and small molecules were determined by ITC. Isolation of the IMPCH domain was achieved to analyse binding to a single active site and elucidate the binding parameters observed for both ATIC domains. The potassium dependence has been defined for the first time as Ka = 796 ± 48μM. The role of the potassium ion has been investigated through the characterization of site directed mutants of the potassium binding site. The thermal stability of ATIC was investigated with DSC and DSF, which suggest that the unfolding landscape of ATIC proceeds initially through the separation of the dimer, and then the IMPCH domain or monomer unfolds at a higher temperature. AICAR, PteGlu4 and KCl all enhance protein stability. The dimerisation of ATIC was investigated by ITC dilution, dimerisation inhibitors, and fluorescent techniques. Unfortunately, none of these techniques were capable of detecting the monomer-dimer equilibrium. However, the dimerisation of ATIC appears to be an entropically-driven event.
Supervisor: Webb, Mike Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.733561  DOI: Not available
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