title: Hydrogen peroxide (H2O2) irreversibly inactivates creatine kinase from Pelodiscus sinensis by targeting the active site cysteine.
abstract: 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 H2O2on PSCKM (muscle type creatine kinase from Pelodiscus sinensis) by the integrating method between enzyme kinetics and docking simulations. We found that H2O2strongly inactivated PSCKM (IC50=0.25mM) in a first-order kinetic process, and targeted the active site cysteine directly. A conformational study showed that H2O2did not induce the tertiary structural changes in PSCKM with no extensive exposure of hydrophobic surfaces. Sequential docking simulations between PSCKM and H2O2indicated that H2O2interacts with the ADP binding region of the active site, consistent with experimental results that demonstrated H2O2-induced inactivation. Our study demonstrates the effect of H2O2on 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.
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea; Department of Nanobiotechnology and Bioinformatics, University of Sciences and Technology, Daejeon 305-350, Republic of Korea.
authors.forename: Jinhyuk
authors.initials: J
authors.lastname: Lee
authors.affiliation: Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Hao
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China; Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, PR China. Electronic address: parkyd@hotmail.com.
authors.forename: Yong-Doo
authors.initials: YD
authors.lastname: Park
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China. Electronic address: qianguoying_wanli@hotmail.com.
authors.forename: Guo-Ying
authors.initials: GY
authors.lastname: Qian
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine; D004789:Enzyme Activation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D012104:Reptiles
chemical_list: D006861:Hydrogen Peroxide; D003402:Creatine Kinase; D003545:Cysteine
keywords: Creatine kinase; H(2)O(2); Inhibition