The effect of Zn2+ on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies

Abstract

We studied the effects of Zn2+ on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn2+ inactivated the activity of PSCK (IC50 =.079 ±.004 mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn2+ induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn2+ directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn2+ bind to PSCK and one Zn2+ is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn 2+ with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn2+ on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.