Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism

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 Mg2+ 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 15N-1H 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 Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) 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.

Doing ‘business as usual’ comes with a cost: evaluating energy cost of maintaining plant intracellular K+ homeostasis under saline conditions

“…The physiological role and decoding mechanisms for these K + flux 'signatures' remain to be elucidated. In mammalian systems, adenosine kinase couples sensing of cellular K + depletion to purine metabolism (de Oliveira et al, 2018). Given the presence of purine receptors in plant systems and their role in signalling (Demidchik et al, 2009) and cell fate determination (Sun et al, 2012), a similar scenario may be potentially envisaged here.…”

Purines: From Diagnostic Biomarkers to Therapeutic Agents in Brain Injury

“…The accumulation of adenosine in the extracellular space occurs rapidly (within seconds) in response to a variety of insults to the mammalian, including human, brain (for a review see [40] ). This accumulation is initiated by ATP depletion, but may be facilitated by the inhibition of adenosine kinase by conditions found during metabolic stress (hypoxia, ATP depletion, ADP, AMP, high levels of intracellular adenosine and low cellular K + ) [49] . This inhibition of the enzyme largely responsible for the inward gradient promoting adenosine uptake, likely contributes to the prolonged presence of extracellular adenosine observed in vitro after oxygen/glucose deprivation, and which has a corresponding inhibitory influence on excitatory synaptic transmission [50] .…”

Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds

“…These compete for a binding site, with the degree of charge on the phosphorus atom directing either activation or inhibition [29]. Several RKs also require a potassium ion in a structural role that is close to the nucleotide binding pocket [76][77][78][79]. These mechanisms are very different from those that the HKs use for control.…”