Activation energy of catalysis-related domain motion in E. coli adenylate kinase

Adenylate kinase from E. coli (AKeco), folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP ↔ 2ADP. Previous X-ray crystallography and optical solution methods showed that the domains AMPbd and LID, and the conserved P-loop, execute large-amplitude catalysis-related motions. We used ¹⁵N NMR spin relaxation methods to find that the simplified model-free (MF) analysis does not, whereas our general Slowly Relaxing Local Structure analysis does, detect catalytic domain motion. SRLS set for the first time the correlation time for domain motion at τ^L_⊥. = 8.2 ns, to be compared with τ_m = 15.1 ns for global tumbling. These results were obtained at 303 K. Herein we conduct a temperature-dependent investigation of τ^L_⊥ and τ_m in the range of 288-310 K. We found that the activation energy for global tumbling is E_a = 16.9 ± 0.5 kJ/mol, the hydrodynamic volume of hydrated AKeco is 65.6 ± 2.1 nm³, its radius is 2.50 ± 0.03 nm, and the number of hydration layers is 1.77. The average τ^L_⊥ value decreases from 11 ns at 288 K to 4 ns at 310 K, with activation energies of 29.7 ± 3.3, 32.1 ± 4.3, and 30.4 ± 4.3 kJ/mol for the domains AMPbd and LID, and the catalytic P-loop, respectively. These values are two-to-three times smaller than typical activation energies of enzymatic reactions. Hence kinase catalysis appears not to be controlled by domain motion in the ligand-free enzyme. However, the latter process clearly facilitates important mechanical aspects such as steric recognition and capturing of the AMP and ATP substrates, their proper positioning for phosphorylation, and the release of the ADP product.