Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism

• Published in: Scientific reports Vol. 8; no. 1; pp. 11988 - 14
• Main Authors: de Oliveira, Renata Rocha, Morales-Neto, Raphael, Rocco, Silvana Aparecida, Sforça, Maurício Luis, Polo, Carla Cristina, Tonoli, Celisa Caldana Costa, Mercaldi, Gustavo Fernando, Cordeiro, Artur Torres, Murakami, Mário Tyago, Franchini, Kleber Gomes
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 10.08.2018; Nature Publishing Group
• Subjects: 140/131; 631/45/535/1266; 631/45/607/275; 82/16; 82/29; 82/6; 82/80; 82/81; 82/83; Adenosine; Adenosine diphosphate; Adenosine kinase; Adenosine Kinase - chemistry; Adenosine Kinase - genetics; Adenosine Kinase - metabolism; Allosteric properties; Amino Acids; ATP; Binding Sites; Enzyme Activation; Humanities and Social Sciences; Intracellular; Kinetics; Magnesium; Magnetic Resonance Imaging; Metabolic Networks and Pathways; Metabolism; Molecular Conformation; multidisciplinary; Mutation; NMR; Nuclear magnetic resonance; Phosphorylation; Potassium; Potassium - metabolism; Protein Binding; Purines - chemistry; Purines - metabolism; Science; Science (multidisciplinary); Structure-Activity Relationship
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-30418-5

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.