Hydrogen peroxide (H₂O₂) irreversibly inactivates creatine kinase from Pelodiscus sinensis by targeting the active...

Creatine kinase (EC 2.7.3.2, CK) plays an important role in cellular energy metabolism and homeostasis by catalysing the transfer of phosphate between ATP and creatine phosphate. In this study, we investigated the effects of H₂O₂ on PSCKM (muscle type creatine kinase from Pelodiscus sinensis) by the integrating method between enzyme kinetics and docking simulations. We found that H₂O₂ strongly inactivated PSCKM (IC₅₀=0.25mM) in a first-order kinetic process, and targeted the active site cysteine directly. A conformational study showed that H₂O₂ did not induce the tertiary structural changes in PSCKM with no extensive exposure of hydrophobic surfaces. Sequential docking simulations between PSCKM and H₂O₂ indicated that H₂O₂ interacts with the ADP binding region of the active site, consistent with experimental results that demonstrated H₂O₂-induced inactivation. Our study demonstrates the effect of H₂O₂ on PSCKM enzymatic function and unfolding, and provides important insight into the changes undergone by this central metabolic enzyme in ectothermic animals in response to the environment.