title: Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon.
abstract: A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb. et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B. p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction. This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8. The enzyme does not sediment at 100,000g. Enzyme activity did not require metal cofactors. The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM). Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism. Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released. The data indicate the formation of a dead-end complex between PHB-glc and the enzyme. Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.
authors.affiliation: Institut für Pharmazeutische Biologie der Rheinischen Friedrich-Wilhelms-Universität Bonn, Federal Republic of Germany.
authors.forename: A
authors.initials: A
authors.lastname: Bechthold
authors.forename: U
authors.initials: U
authors.lastname: Berger
authors.forename: L
authors.initials: L
authors.lastname: Heide
mesh_terms: D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005964:Glucosyltransferases; C070984:4-hydroxybenzoate 4-O-glucosyltransferase