title: Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction.
abstract: Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into UDP-glucuronic acid. The catalytic reaction proceeds in two NAD(+)-dependent steps via covalent thiohemiacetal and thioester enzyme intermediates. Formation of the thiohemiacetal adduct occurs through attack of Cys(276) on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because previous studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting Glu(161)→Gln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measurements showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transformations. The product contained no deuterium at C-5 within the detection limit (≤2%). The results are consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is immediately trapped by thiohemiacetal adduct formation.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eixelsberger
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D055162:Biocatalysis; D003903:Deuterium; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D014532:Uridine Diphosphate Glucose / Q000737:chemistry* / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D014867:Water; D009243:NAD; D014535:Uridine Diphosphate Glucuronic Acid; D003903:Deuterium; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014532:Uridine Diphosphate Glucose