The effect of Zn(2+) on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies.
We studied the effects of Zn(2+) on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn(2+) inactivated the activity of PSCK (IC(50) = .079 ± .004 mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn(2+) induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn(2+) 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 Zn(2+) bind to PSCK and one Zn(2+) 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 Zn(2+) 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.
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, 315100, PR China.
Su-Fang
SF
Wang
Jinhyuk
J
Lee
Wei
W
Wang
Yue-Xiu
YX
Si
Caiyan
C
Li
Tae-Rae
TR
Kim
Jun-Mo
JM
Yang
Shang-Jun
SJ
Yin
Guo-Ying
GY
Qian
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D017510:Protein Folding; D016415:Sequence Alignment; D014426:Turtles / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
D018076:DNA, Complementary; D003402:Creatine Kinase; D015032:Zinc; D005998:Glycine