Adenylate kinases of thermophiles Aquifex aeolicus and Geobacillus stearothermophilus: biochemical and kinetic studies

Adenylate kinases (AK) play a pivotal role in the regulation of cellular energy. The aim of our work was to achieve the overproduction and purification of AKs from two groups of bacteria and to determine, for the first time, the comprehensive biochemical and kinetic properties of adenylate kinase from Gram-negative Aquifex aeolicus (AKaq) and Gram-positive Geobacillus stearothermophilus (AKst). Therefore we determined KM and Vmax values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor Ap5A for both thermophilic AKs. The kinetic studies indicate that both AKs exhibit significantly higher affinity for substrates with the pyrophosphate group than for adenosine monophosphate. AK activation by Mg2+ and Mn2+ revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, Mn2+ ions at 0.2–2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than Mg2+ ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while Ap5A at a concentration of 10 µmol/L and 50 µmol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by Mg2+, suggesting the allosteric effect of ATP for both bacterial AKs.

Crystal structure of adenylate kinase from an extremophilic archaeon Aeropyrum pernix with ATP and AMP

The crystal structure of an adenylate kinase from an extremophilic archaeon Aeropyrum pernix was determined in complex with full ligands, ATP-Mg2+ and AMP, at a resolution of 2.0 Å. The protein forms a trimer as found for other adenylate kinases from archaea. Interestingly, the reacting three atoms, two phosphorus and one oxygen atoms, were located almost in line, supporting the SN2 nucleophilic substitution reaction mechanism. Based on the crystal structure obtained, the reaction coordinate was estimated by the quantum mechanics calculations combined with molecular dynamics. It was found that the reaction undergoes two energy barriers; the steps for breaking the bond between the oxygen and γ-phosphorus atoms of ATP to produce a phosphoryl fragment and creating the bond between the phosphoryl fragment and the oxygen atom of the β-phosphate group of ADP. The reaction coordinate analysis also suggested the role of amino-acid residues for the catalysis of adenylate kinase.

The inhibition of adenylate kinase by 2,4-thiazolidinedione evaluated by protein-ligand docking

<p class="ADMETabstracttext">Due to its crucial role in nucleotide metabolism, adenylate kinase deserves a special attention in screening of potential inhibitors. Herein, we report the assessment of the relative orientation of the ligand 2,4-thiazolidinedione to adenylate kinase crystallized in closed conformation. Protein-ligand docking was performed to estimate the binding energy and inhibition constant of 2,4-thiazolidinedione to the adenylate kinases’ active sites from different organisms. Our results revealed the best orientation of 2,4-thiazolidinedione is with Gram-positive and acid fast bacteria adenylate kinase – K_i = 0.76±0.1 mM and binding energy -4.26±0.08 kcal/mol. Human adenylate kinases display unfavourable interactions, the binding affinity fluctuating among K_i=0.84 mM and 8.8 mM (3.88±3.51); the energy binding -3.56±0.57. From the three human adenylate kinases analysed, only isoenzyme 2 shows a binding conformation similar to its counterpart from E. coli. Adenylate kinase - this small enzyme needed for survival of every organisms - interacts differently with 2,4-thiazolidinedione, this selectivity being the most important evidence of the present study.</p>