title: Towards Binding Mechanism of Cu2+ on Creatine Kinase from Pelodiscus sinensis: Molecular Dynamics Simulation Integrating Inhibition Kinetics Study.
abstract: BACKGROUND
  Cu2+ is well known to play important roles in living organisms having bifacial distinction: essential microelement that is necessary for a wide range of metabolic processes but hyper-accumulation of Cu2+ can be toxic. The physiological function of Cu2+ in ectothermic animals such as Pelodiscus sinensis (Chinese soft-shelled turtle) has not been elucidated.
  
  
  OBJECTIVE
  In this study, we elucidated effect of Cu2+ on the energy producing metabolic enzyme creatine kinase (CK), which might directly affect energy metabolism and homeostasis of P. sinensis.
  
  
  METHODS
  We first conducted molecular dynamics (MD) simulations between P-CK and Cu2+ and conducted the inactivation kinetics including spectrofluorimetry study.
  
  
  RESULTS
  MD simulation showed that Cu2+ blocked the binding site of the ATP cofactor, indicating that Cu2+ could directly inactivate P-CK. We prepared the muscle type of CK (P-CK) and confirmed that Cu2+ conspicuously inactivated the activity of P-CK (IC50 = 24.3 μM) and exhibited non-competitive inhibition manner with creatine and ATP in a first-order kinetic process. This result was well matched to the MD simulation results that Cu2+-induced non-competitive inactivation of P-CK. The spectrofluorimetry study revealed that Cu2+ induced tertiary structure changes in PCK accompanying with the exposure of hydrophobic surfaces. Interestingly, the addition of osmolytes (glycine, proline, and liquaemin) effectively restored activity of the Cu2+-inactivated P-CK.
  
  
  CONCLUSIONS
  Our study illustrates the Cu2+-mediated unfolding of P-CK with disruption of the enzymatic function and the protective restoration role of osmolytes on P-CK inactivation. This study provides information of interest on P-CK as a metabolic enzyme of ectothermic animal in response to Cu2+ binding.
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806. Korea.
authors.forename: Jinhyuk
authors.initials: J
authors.lastname: Lee
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China.
authors.forename: Yong-Doo
authors.initials: YD
authors.lastname: Park
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China.
authors.forename: Guo-Ying
authors.initials: GY
authors.lastname: Qian
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D003401:Creatine / Q000737:chemistry; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D017510:Protein Folding; D014426:Turtles / Q000235:genetics*
chemical_list: D003300:Copper; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D003401:Creatine
keywords: Creatine kinase; Cu2+; MD simulation; Pelodiscus sinensis; inactivation; osmolytes