title: Effects of cadmium on the cuttlefish Sepia pharaonis' arginine kinase: unfolding kinetics integrated with computational simulations.
abstract: Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.
authors.affiliation: a School of Marine Sciences , Ningbo University , Ningbo 315211 , P.R. China.|b College of Biological and Environmental Sciences, Zhejiang Wanli University , Ningbo 315100 , P.R. China.
authors.forename: Yue-Xiu
authors.initials: YX
authors.lastname: Si
authors.affiliation: c Korean Bioinformation Center (KOBIC) , Korea Research Institute of Bioscience and Biotechnology , Daejeon 305-806 , Korea.|d Department of Nanobiotechnology and Bioinformatics , University of Sciences and Technology , Daejeon 305-350 , Korea.
authors.forename: Jinhyuk
authors.initials: J
authors.lastname: Lee
authors.affiliation: b College of Biological and Environmental Sciences, Zhejiang Wanli University , Ningbo 315100 , P.R. China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhao
authors.affiliation: b College of Biological and Environmental Sciences, Zhejiang Wanli University , Ningbo 315100 , P.R. China.
authors.forename: Shang-Jun
authors.initials: SJ
authors.lastname: Yin
authors.affiliation: b College of Biological and Environmental Sciences, Zhejiang Wanli University , Ningbo 315100 , P.R. China.|e Zhejiang Provincial Key Laboratory of Applied Enzymology , Yangtze Delta Region Institute of Tsinghua University , Jiaxing 314006 , P.R. China.
authors.forename: Yong-Doo
authors.initials: YD
authors.lastname: Park
authors.affiliation: b College of Biological and Environmental Sciences, Zhejiang Wanli University , Ningbo 315100 , P.R. China.
authors.forename: Guo-Ying
authors.initials: GY
authors.lastname: Qian
authors.affiliation: a School of Marine Sciences , Ningbo University , Ningbo 315211 , P.R. China.
authors.forename: Xia-Min
authors.initials: XM
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry* / Q000633:toxicity; D049832:Decapodiformes / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D009994:Osmolar Concentration; D066329:Protein Aggregates / Q000187:drug effects; D011485:Protein Binding; D017510:Protein Folding; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D066329:Protein Aggregates; D002104:Cadmium; D001122:Arginine Kinase
keywords: MD simulation; arginine kinase; cadmium; cuttlefish; inhibition; osmolytes