Yan Cai, Jinhyuk Lee, Wei Wang, Yong-Doo Park* and Guo-Ying Qian* Pages 534 - 544 ( 11 )
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. Method: 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. Conclusion: 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.
Creatine kinase, Pelodiscus sinensis, Cu2+, MD simulation, inactivation, osmolytes.
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100