Title:Towards Binding Mechanism of Cu2+ on Creatine Kinase from Pelodiscus sinensis: Molecular Dynamics Simulation Integrating Inhibition Kinetics Study
Volume: 24
Issue: 6
Author(s): Yan Cai, Jinhyuk Lee, Wei Wang, Yong-Doo Park*Guo-Ying Qian*
Affiliation:
- College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100,China
- College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100,China
Keywords:
Creatine kinase, Pelodiscus sinensis, Cu2+, MD simulation, inactivation, osmolytes.
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 Cu
2+ 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 Cu
2+ and
conducted the inactivation kinetics including spectrofluorimetry study.
Results: MD simulation showed that Cu
2+blocked the binding site of the ATP cofactor, indicating
that Cu
2+ could directly inactivate P-CK. We prepared the muscle type of CK (P-CK) and confirmed
that Cu
2+ conspicuously inactivated the activity of P-CK (IC
50 = 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 Cu
2+-induced non-competitive inactivation
of P-CK. The spectrofluorimetry study revealed that Cu
2+ 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 Cu
2+-inactivated P-CK.
Conclusion: Our study illustrates the Cu
2+-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 Cu
2+ binding.
