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dc.contributor.authorMcLeod, Matthew 14:54:28 (GMT) 14:54:28 (GMT)
dc.description.abstractPhosphoenolpyruvate carboxykinase (PEPCK) is the metabolic enzyme that catalyzes the first committed step in gluconeogenesis and glyceroneogensis. More recently, PEPCK has been implicated in the maintenance and establishment of infections in macrophages by Mycobacterium tuberculosis, cancer cell growth and metabolic homeostasis, and overall TCA cycle flux as a moderator through removal of cycle anions. Many isoforms of PEPCK have been structurally and kinetically characterized against many different inhibitors. Of these inhibitors, 3-mercaptopincolinic acid (3-MPA) was shown to bind allosterically (Ki ~ 150µM) in an allosteric cleft behind the nucleotide binding pocket, as well competitively in the OAA/PEP binding site (Ki ~ 5µM). A new inhibitor, 3-carboxymethylthio-picolinic acid (CMP), has been designed based on of the structure of 3-MPA and other inhibitory studies by Stiffin et al (2009) in order to create a more selective inhibitor for PEPCK’s active site. Previous studies mapping the active site of GTP-dependent PEPCKs has shown an overlapping secondary binding site adjacent to the competitive binding cleft. CMP has been designed from the previous 3-MPA molecule scaffold but with a simple carboxymethyl tail modification to simultaneously occupy the overlapping site. This study will investigate the structural and kinetic repercussions of the binding of CMP to three isozymes of PEPCK; PEPCK from Mycobacterium tuberculosis, rat cytosolic PEPCK, and finally human mitochondrial PEPCK. The data from the structural crystallographic studies of CMP and rat cytosolic PEPCK, it is shown that CMP does in fact occupy both the competitive site in coordination with the manganese ion but also extends into the overlapping adjacent pocket as intended. The kinetic results of CMP against the three isozymes in the catalytic direction of PEP production shows an Ki ~ 30-90uM. These two results furthermore show that the adjacent binding cleft can be utilized for future, more selective, inhibition of GTP-dependent PEPCK.en
dc.publisherUniversity of Waterlooen
dc.subjectPhosphoenolpyruvate carboxykinaseen
dc.titleInhibition of Phosphoenolpyruvate Carboxykinase with a 3-Mercaptopicolinic acid Analogueen
dc.typeMaster Thesisen
dc.pendingfalse of Waterlooen
uws-etd.degreeMaster of Scienceen
uws.contributor.advisorHolyoak, Todd
uws.contributor.affiliation1Faculty of Scienceen

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