Role of monovalent and divalent metal cations in human ribokinase catalysis and regulation.
Human ribokinase (RK) is a member of the ribokinase family, and is the first enzyme responsible for D-ribose metabolism, since D-ribose must first be converted into D-ribose-5-phosphate to be further metabolized and incorporated into ATP or other high energy phosphorylated compounds. Despite its biological importance, RK is poorly characterized in eukaryotes and especially in human. We have conducted a comprehensive study involving catalytic and regulatory features of the human enzyme, focusing on divalent and monovalent metal regulatory effects. Mg(2+), Mn(2+), and Co(2+) support enzyme activity although at different rates, with Mn(2+) being the most effective. Analysis of the divalent cation requirement in the wild type enzyme demonstrates that in addition to that chelated by the nucleotide substrate, an activating cation (either Mn(2+) or Mg(2+)) is required to obtain full activity of the enzyme, with the affinity for both divalent cations being almost the same (4 and 8 µM respectively). Besides metal cation activation, inhibition of the enzyme activity by increasing concentrations of Mn(2+) but not Mg(2+) is observed. Also the role of residues N199 and E202 of the highly conserved NXXE motif present at the active site has been evaluated regarding Mg(2+) and phosphate binding. K(+) (but not Na(+)) and PO4 (3-) activate the wild type enzyme, whereas the N199L and E202L mutants display a dramatic decrease in kcat and require higher free Mg(2+) concentrations than the wild type enzyme to reach maximal activity, and the activating effect of PO4 (3-) is lost. The results demonstrated a complex regulation of the human ribokinase activity where residues Asn199 and Glu202 play an important role.
Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
Diego
D
Quiroga-Roger
Jorge
J
Babul
Victoria
V
Guixé
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D055162:Biocatalysis; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D011189:Potassium Chloride / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry
D010710:Phosphates; D017680:Potassium Compounds; D008345:Manganese; D012965:Sodium Chloride; D011189:Potassium Chloride; C013216:potassium phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022534:ribokinase; D008274:Magnesium; C018279:sodium phosphate