Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism.

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D N-H HSQC experiments revealed that the binding of K perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent K = 10.4 mM). Mechanistically, K increases the catalytic turnover (K) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K and highlights the role of ADK in connecting depletion of intracellular K to the regulation of purine metabolism.