pH-dependent structural dynamics and catalytic function of human thymidine kinase 1

Human thymidine kinase 1 (hTK1) is a key enzyme in the thymidine salvage pathway that regulates DNA synthesis and repair and serves as an important biomarker of cellular proliferation and cancer. However, the influence of pH on its structural stability and catalytic function remains poorly understood. In this study, the conformational and functional behavior of recombinant hTK1 was systematically investigated using a combination of experimental and computational approaches. We observed that hTK1 retained its highest structural stability and catalytic activity at pH 8.0, exhibiting the greatest Vmax (1.363 ± 0.06 μM s^-1) and 100% relative activity. ANS fluorescence increased with lower pH, while ThT fluorescence, light scattering, and dynamic light scattering showed stronger signals associated with aggregation at higher pH. MD simulations and spectroscopic data showed that under extreme pH conditions, the structures were more flexible, hydrogen-bonding networks changed, solvent accessibility increased, and correlated residue motions were disrupted. The pH 8.0 condition was characterized by a single conformational cluster of hTK1, whereas the acidic and alkaline conditions were more conformationally heterogeneous. These structural perturbations substantially reduced kinase activity, strongly suggesting a relationship between protein conformation and kinase activity. The results show that the native conformation is crucial for maximal catalytic activity and provide molecular insights into hTK1 regulation under physiological and pathological conditions. We combine experimental characterization and MD simulations to probe the link between pH-dependent structural dynamics and hTK1 function, further strengthening hTK1's potential as a therapeutic target to cure diseases associated with altered nucleotide metabolism.