title: Ribokinase from E. coli: expression, purification, and substrate specificity.
abstract: Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 micromol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium>>>ammonium>cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides D-ribose and 2-deoxy-D-ribose, RK was found to catalyze the 5-O-phosphorylation of D-arabinose, D-xylose, and D-fructose in the presence of ATP, and potassium and magnesium ions; L-ribose and L-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of D-pentofuranose-5-[32P]phosphates in the presence of [gamma-32P]ATP and RK is reported.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Chuvikovsky
authors.forename: Roman S
authors.initials: RS
authors.lastname: Esipov
authors.forename: Yuri S
authors.initials: YS
authors.lastname: Skoblov
authors.forename: Larisa A
authors.initials: LA
authors.lastname: Chupova
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Muravyova
authors.forename: Anatoly I
authors.initials: AI
authors.lastname: Miroshnikov
authors.forename: Seppo
authors.initials: S
authors.lastname: Lapinjoki
authors.forename: Igor A
authors.initials: IA
authors.lastname: Mikhailopulo
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009705:Nucleosides / Q000138:chemical synthesis / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D009705:Nucleosides; D017853:Phosphotransferases (Alcohol Group Acceptor); C022534:ribokinase