title: Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase.
abstract: UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.
authors.affiliation: Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 90187 Umeå, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Decker
authors.forename: Meng
authors.initials: M
authors.lastname: Meng
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Gornicka
authors.forename: Anders
authors.initials: A
authors.lastname: Hofer
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Wilczynska
authors.forename: Leszek A
authors.initials: LA
authors.lastname: Kleczkowski
mesh_terms: D005695:Galactosephosphates / Q000378:metabolism / Q000494:pharmacology; D005958:Glucosephosphates / Q000378:metabolism / Q000494:pharmacology; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000378:metabolism / Q000494:pharmacology
chemical_list: D005695:Galactosephosphates; D005958:Glucosephosphates; C029973:galactose-1-phosphate; C031590:glucose-1-phosphate; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014544:Uridine Triphosphate; D014532:Uridine Diphosphate Glucose