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Hysteresis and Allostery in Human UDP-Glucose Dehydrogenase Require a Flexible Protein Core.

Authors
  • Beattie, Nathaniel R1
  • Pioso, Brittany J1
  • Sidlo, Andrew M1
  • Keul, Nicholas D1
  • Wood, Zachary A1
  • 1 Department of Biochemistry & Molecular Biology , University of Georgia , Athens , Georgia 30602 , United States. , (Georgia)
Type
Published Article
Journal
Biochemistry
Publisher
American Chemical Society
Publication Date
Dec 18, 2018
Volume
57
Issue
50
Pages
6848–6859
Identifiers
DOI: 10.1021/acs.biochem.8b00497
PMID: 30457329
Source
Medline
Language
English
License
Unknown

Abstract

Human UDP-glucose dehydrogenase (hUGDH) oxidizes UDP-glucose to UDP-glucuronic acid, an essential substrate in the phase II metabolism of drugs. The activity of hUGDH is regulated by the conformation of a buried allosteric switch (T131 loop/α6 helix). Substrate binding induces the allosteric switch to slowly isomerize from an inactive E* conformation to the active E state, which can be observed as enzyme hysteresis. When the feedback inhibitor UDP-xylose binds, the allosteric switch and surrounding residues in the protein core repack, converting the hexamer into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition is facilitated by large cavities and declivities in the protein core that provide the space required to accommodate the alternate packing arrangements. Here, we have used the A104L substitution to fill a cavity in the E state and sterically prevent repacking of the core into the EΩ state. Steady state analysis shows that hUGDHA104L binds UDP-xylose with lower affinity and that the inhibition is no longer cooperative. This means that the allosteric transition to the high-UDP-xylose affinity EΩ state is blocked by the substitution. The crystal structures of hUGDHA104L show that the allosteric switch still adopts the E and E* states, albeit with a more rigid protein core. However, the progress curves of hUGDHA104L do not show hysteresis, which suggests that the E* and E states are now in rapid equilibrium. Our data suggest that hysteresis in native hUGDH originates from the conformational entropy of the E* state protein core.

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