Pentavalent Ions Dependency Is a Conserved Property of Adenosine Kinase from Diverse Sources: Identification of a Novel Motif Implicated in Phosphate and Magnesium Ion Binding and Substrate Inhibition †

The catalytic activity of adenosine kinase (AK) from mammalian sources has previously been shown to exhibit a marked dependency upon the presence of pentavalent ions (PVI), such as phosphate (PO 4 ), arsenate, or vanadate.We now show that the activity of AK from diverse sources, including plant, yeast, and protist species, is also markedly enhanced in the presence of PVI. In all cases, PO 4 or other PVI exerted their effects primarily by decreasing the K m for adenosine and alleviating the inhibition caused by high concentrations of substrates. These results provide evidence that PVI dependency is a conserved property of AK and perhaps of the PfkB family of carbohydrate kinases which includes AK. On the basis of sequence alignments, we have identified a conserved motif NXXE within the PfkB family. The N and E of this motif make close contacts with Mg 2 + and PO 4 ions in the crystal structures of AK and bacterial ribokinase (another PfkB member which shows PVI dependency), implicating these residues in their binding. Site-directed mutagenesis of these residues in Chinese hamster AK have resulted in active proteins with greatly altered phosphate stimulation and substrate inhibition characteristics. The N239Q mutation leads to the formation of an active protein whose activity was not stimulated by PO 4 or inhibited by high concentrations of adenosine or ATP. The activity of the E242D mutant protein was also not significantly altered in the presence of phosphate. Although PO 4 had no effect on the K m A d e n o s i n e for this mutant, the K m A T P , K i A d e n o s i n e , and K i A T P were significantly decreased. In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg 2 + requirement. These observations support the view that N239 and E242 play an important role in the binding of PO 4 and Mg 2 + ions required for the catalytic activity of adenosine kinase.