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

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 HO on PSCKM (muscle type creatine kinase from Pelodiscus sinensis) by the integrating method between enzyme kinetics and docking simulations. We found that HO strongly inactivated PSCKM (IC=0.25mM) in a first-order kinetic process, and targeted the active site cysteine directly. A conformational study showed that HO did not induce the tertiary structural changes in PSCKM with no extensive exposure of hydrophobic surfaces. Sequential docking simulations between PSCKM and HO indicated that HO interacts with the ADP binding region of the active site, consistent with experimental results that demonstrated HO-induced inactivation. Our study demonstrates the effect of HO 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.