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Title: The molecular basis of negative cooperativity : a biochemical study of NAD (P)H-quinone oxidoreductases
Author: Megarity, Clare Frances
ISNI:       0000 0004 5371 6504
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2014
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Negative cooperativity refers to the mechanism whereby the sequential binding of ligands to a protein decreases in affinity. A protein's native state ensemble consists of numerous conformers and is dynamic. A ligand which induces negative cooperativity causes a shift in the distribution of this ensemble such that the subsequent molecules bind with less affinity; this requires a physically linked pathway through which the change is propagated. Positive cooperativity has been well documented but the mechanisms and advantages of negative cooperativity are less understood. To this end, the research presented here focuses on the mechanism of negative cooperativity in human NAD(P)H quinone oxidoreductase 1 (NQ01), human NRH quinone oxidoreductase (NQ02), MdaB from Escherichia coli and Lot6p from Saccharomyces cerevisiae. This thesis presents evidence to support the presence of a pathway linking the active sites in NQ01, through which, negative cooperativity towards the inhibitor dicoumarol propagates. Glycine 151 within this pathway has been identified as pivotal to this mechanism. Two cancer-associated variants of NQ01, p.P187S and p.R139W, have been characterised. Neither change affected negative cooperativity; the proline to serine change was the most severe in terms of structure and function. Two variants of human NQ02 have been compared. NQ02-47L is overall more flexible and exists as an ensemble containing three metastable states; NQ02-47F exhibits negative cooperativity. MdaB has been characterised and novel inhibitors identified. An alteration which decreased steric hindrance in the active site allowed for enhanced dicoumarol inhibition; however, unlike NQ01 , MdaB did not exhibit negative cooperativity towards this inhibitor. An additional alteration to increase flexibility did not enable the enzyme to exhibit negative cooperativity. Lot6p has been characterised and novel inhibitors identified some of which, induced negative cooperativity. An interconnecting pathway and a specific glycine within it have been identified as central to the mechanism of negative cooperativity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available