Abstract
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(2+) 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 (15)N-(1)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(d) = 10.4 mM). Mechanistically, K(+) increases the catalytic turnover (K(cat)) 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.