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dc.contributor.authorEdwards, Achelle A.
dc.date.accessioned2018-07-12T17:35:45Z
dc.date.available2018-07-12T17:35:45Z
dc.date.issued2009
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 70-05, Section: B, page: 2902.;Advisors: Vern L. Schramm.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3360938
dc.identifier.urihttps://hdl.handle.net/20.500.12202/1055
dc.description.abstractPurine nucleoside phosphorylases (PNPs) catalyze the reversible interconversion of (deoxy)nucleosides to free purine base and (deoxy)ribose 1-phosphate. The Immucillins are potent transition state analogue inhibitors of PNPs, many with picomolar dissociation constants and one being in clinical trial for the treatment of T-cell proliferative disorders. The purpose of this thermodynamic investigation is to define the source of favorable binding affinities for the Immucillins in terms of enthalpy (DeltaH), entropy (-TDeltaS) and dynamic contributions towards the binding energy (DeltaG).;Isothermal titration calorimetry (ITC) allowed a full thermodynamic investigation of the PNPs. Binding the Immucillins to a single subunit of human PNP revealed that they bind with favorable binding energy that is enthalpy-driven with minor entropic penalties. Binding the Immucillins to all three sites of human PNP revealed that binding occurs with negative cooperativity evidenced by reduction in binding affinities, less exothermic enthalpy changes and more favorable entropy changes as each site fills.;Entropy changes for binding the Immucillins to human PNP are interpreted in terms of protein conformational changes that occur as the inhibitor binds. Hydrogen-deuterium exchange, analytical ultracentrifugation and thermal shift assays revealed that Immucillin binding to human PNP causes reduced dynamics and increased protein 'packing', consistent with the entropy results from single subunit titrations, as well as increased thermal stability.;Thermodynamic comparison of the Immucillins, a substrate analogue (9-deazainosine) and hypoxanthine binding to human PNP revealed that the Immucillins bind with negative cooperativity but 9-deazainosine and hypoxanthine bind equally to all sites with binding dominated by highly exothermic enthalpy changes and large unfavorable entropy changes.;Catalytic sites of human PNP are at the subunit interface with Phe159 being contributed from the adjacent subunit. The Phe159Gly mutant of human PNP results in loss of negative cooperative binding of the Immucillins demonstrating the structural link for negative cooperativity. Thermodynamic investigations with PNP from Plasmodium falciparum demonstrated that Immucillins bind to this hexameric enzyme with equal affinity to all sites resulting in binding that is promoted by large exothermic enthalpy changes and entropy changes that are unfavorable.
dc.publisherProQuest Dissertations & Theses
dc.subjectBiochemistry.
dc.titleThermodynamics of binding to purine nucleoside phosphorylases
dc.typeDissertation


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