Role of monovalent and divalent metal cations in human ribokinase catalysis and regulation

Abstract

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. Mg²⁺, Mn²⁺, and Co²⁺ support enzyme activity although at different rates, with Mn²⁺ 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 Mn²⁺ or Mg²⁺) 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 Mn²⁺ but not Mg²⁺ 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 Mg²⁺ 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 k_cat and require higher free Mg²⁺ 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.