Show simple item record

dc.contributor.authorMcCluskey, Gregory
dc.date.accessioned2019-11-28T15:06:48Z
dc.date.available2019-11-28T15:06:48Z
dc.identifier.urihttp://hdl.handle.net/10222/76668
dc.description.abstractCytidine-5′-triphosphate (CTP) synthase (CTPS) catalyzes the biosynthesis of CTP from UTP using either L-glutamine or free NH3 as a substrate. Glutamine is hydrolyzed in the glutamine amidotransferase (GATase) domain, with GTP acting as a positive allosteric effector, and nascent NH3 is transported through a ~25-Å tunnel to the synthase domain where CTP is generated. The requirement for CTP in cell growth and proliferation has highlighted the importance of CTPS in a variety of infectious diseases, cancer, and the immune response. Consequently, CTPS is a recognized target for the development of chemotherapeutic agents. To better understand the inhibition of CTPS by the drug metabolite gemcitabine- 5′-triphosphate (dF-dCTP), we used the Escherichia coli variant (EcCTPS) as a model because of its ease of purification and similarities to human CTPS. dF-dCTP was a parabolic competitive inhibitor (Ki = 3.0 ± 0.1 μM) that likely bound tightly through interactions between the 2′-arabino fluorine and an interdigitating loop in the CTP-binding site. Inhibition by dF-dCTP, but not enzymatic activity with UTP, was ablated by mutation of Glu 149 to Asp (E149D). This variant could recycle dF-dUTP, the 'inactive' catabolite of dF-dCTP, which may have consequences for gemcitabine pharmacology in vivo. Using dynamic light scattering and transmission electron microscopy, we showed that dF-dCTP induces EcCTPS filament formation, and disassembly was facilitated by UTP. E149D and variants of Phe 227 (F227A and F227L) were unable to undergo large-scale filament formation, supporting the role of Phe 227 as a 'sensor' of CTP that promotes filament formation. Molecular gates within enzymes often play important roles in synchronizing catalytic events. The determinants of NH3 channelling through a putative gate were investigated by site-directed mutagenesis. Point mutations at Val 60 (V60A, V60C, V60D, V60W, and V60F), at the most constricted point of the NH3 tunnel, had varying consequences on catalysis. Notably, the V60F variant could not utilize exogenous NH3 as a substrate, but permitted passage of glutamine-derived NH3. Addition of Gln, or a combination of GTP with modification of V60F with 6-diazo-5-oxo-L-norleucine, enhanced NH3 channelling, suggesting that the GATase activity promoted opening of the gate.en_US
dc.language.isoenen_US
dc.subjectdynamic light scatteringen_US
dc.subjecttransmission electron microscopyen_US
dc.subjectenzyme kineticsen_US
dc.titleINVESTIGATION OF INHIBITION, FILAMENT FORMATION, AND INTER-DOMAIN AMMONIA CHANNELLING OF CTP SYNTHASEen_US
dc.date.defence2018-03-22
dc.contributor.departmentDepartment of Biochemistry & Molecular Biologyen_US
dc.contributor.degreeDoctor of Philosophyen_US
dc.contributor.external-examinerDr. Andrew Gulicken_US
dc.contributor.graduate-coordinatorDr. Jan Raineyen_US
dc.contributor.thesis-readerDr. David Langelaanen_US
dc.contributor.thesis-readerDr. Vanya Ewarten_US
dc.contributor.thesis-readerDr. Jan Raineyen_US
dc.contributor.thesis-supervisorDr. Stephen Bearneen_US
dc.contributor.ethics-approvalNot Applicableen_US
dc.contributor.manuscriptsNot Applicableen_US
dc.contributor.copyright-releaseYesen_US
 Find Full text

Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record