title: Hysteresis in human UDP-glucose dehydrogenase is due to a restrained hexameric structure that favors feedback inhibition.
abstract: Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Renuka
authors.initials: R
authors.lastname: Kadirvelraj
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Custer
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Keul
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Sennett
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Sidlo
authors.forename: Richard M
authors.initials: RM
authors.lastname: Walsh
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Wood
mesh_terms: D000494:Allosteric Regulation; D001667:Binding, Competitive; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D025461:Feedback, Physiological; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism / Q000494:pharmacology
chemical_list: D007527:Isoenzymes; D008024:Ligands; D011994:Recombinant Proteins; D014540:Uridine Diphosphate Xylose; D014533:Uridine Diphosphate Glucose Dehydrogenase