Ribokinase from E. coli: Expression, purification, and substrate specificity

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 μmol/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 [γ-32P]ATP and RK is reported.

Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells, purified from sonicated cells by double chromatography to afford a preparation with specific activity of 75 μmol/min mg protein. 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-ribofuranose-5-[32P]phosphate in the presence of [γ-32P]ATP and RK is reported.Figure optionsDownload as PowerPoint slide