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dc.contributor.authorSennett, Nicholas Cody
dc.date.accessioned2014-03-04T21:04:00Z
dc.date.available2014-03-04T21:04:00Z
dc.date.issued2013-05
dc.identifier.othersennett_nicholas_c_201305_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/sennett_nicholas_c_201305_phd
dc.identifier.urihttp://hdl.handle.net/10724/28896
dc.description.abstractHuman UDP-α-D-glucose dehydrogenase (hUGDH) catalyzes the oxidation of UDP-glucose (UDG) to UDP-glucuronic acid (UGA) using two equivalents of NAD+. hUGDH is regulated by the product of another enzyme, UDP-xylose (UDX). We have shown that UDX is a heterotropic allosteric effector that binds to the active site of hUGDH. UDX stabilizes an unusual horseshoe-shaped hexameric state that differs from the 32 symmetry hexamers observed in substrate bound structures. The primary difference between these structures is the translation of a loop in the active site (Thr131-loop) by ~5Å that changes packing interactions in the protein core and a rotation of a helix (α6136-144) located in the hexamer interface. But how does the Thr131-loop change conformations given the steric packing constraints of the protein core? To answer this question, we deleted Val132 from the Thr131-loop to approximate an intermediate state. The 2.3 Å resolution crystal structure of the deletion construct (Δ132) reveals an open conformation 32 symmetry hexamer that relaxes steric constraints and facilitates repacking of the protein core. In addition, the structure of the Δ132 construct has a hexamer interface similar to the UDX- inhibited enzyme suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states. While UDX stabilizes a horseshoe shaped hexamer our sedimentation velocity studies show that the unliganded enzyme is a mixture of dimers, tetramers and hexamers. To investigate the role of the 32 symmetry hexamer in the catalytic cycle, we engineered a stable hUGDH dimer by introducing a charge-switch substitution (K94E) in the hexamer-building interface (hUGDHK94E). The kcat of hUGDHK94E is ∼160-fold lower than that of the wild-type enzyme, suggesting that the hexamer is the catalytically relevant state. We also show that cofactor binding triggers the formation of the 32 symmetry hexamer. The hUGDHK94E crystal structure at 2.08 Å resolution identifies loop88−110 as the cofactor-responsive allosteric switch that drives hexamer formation; loop88−110 directly links cofactor binding to the stability of the hexamer-building interface.
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjectUDP-Glucose Dehydrogenase, UGDH, Allostery, Allokleidy, Feedback Inhibition, Oxidoreductase, Oligomeric State, Molecular Switch
dc.titleThe allosteric regulation of human UDP-α-D-glucose-6-dehydrogenase
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorZachary Wood
dc.description.committeeZachary Wood
dc.description.committeeJames Prestegard
dc.description.committeeKelley Moremen
dc.description.committeeWilliam Lanzilotta


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