title: Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.
abstract: The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.
authors.affiliation: Department of Biochemistry, University of Nebraska , Lincoln, Nebraska 68588-0664, United States.
authors.forename: George
authors.initials: G
authors.lastname: Grady
authors.affiliation: Department of Biochemistry, University of Nebraska , Lincoln, Nebraska 68588-0664, United States.
authors.forename: Ashley
authors.initials: A
authors.lastname: Thelen
authors.affiliation: Department of Biochemistry, University of Nebraska , Lincoln, Nebraska 68588-0664, United States.
authors.forename: Jaleen
authors.initials: J
authors.lastname: Albers
authors.affiliation: Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588-0304, United States.
authors.forename: Tong
authors.initials: T
authors.lastname: Ju
authors.affiliation: Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588-0304, United States.
authors.forename: Jiantao
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Biochemistry, University of Nebraska , Lincoln, Nebraska 68588-0664, United States.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Barycki
authors.affiliation: Department of Biochemistry, University of Nebraska , Lincoln, Nebraska 68588-0664, United States.
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Simpson
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000528:radiation effects; D002384:Catalysis; D003432:Cross-Linking Reagents; D006801:Humans; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D010084:Oxidation-Reduction; D055668:Photochemical Processes; D011487:Protein Conformation; D055503:Protein Multimerization / Q000528:radiation effects*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014532:Uridine Diphosphate Glucose