The Inhibitory Effects of Cu(2+) on Exopalaemon carinicauda Arginine Kinase via Inhibition Kinetics and Molecular Dynamics Simulations.
We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 μM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, 315100, People's Republic of China.
Yue-Xiu
YX
Si
Jinhyuk
J
Lee
Shang-Jun
SJ
Yin
Xiao-Xu
XX
Gu
Yong-Doo
YD
Park
Guo-Ying
GY
Qian
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001122:Arginine Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D060829:Arthropod Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D020134:Catalytic Domain; D002413:Cations, Divalent; D003300:Copper / Q000737:chemistry*; D033364:Decapoda / Q000737:chemistry* / Q000201:enzymology; D005998:Glycine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry; D066329:Protein Aggregates; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary
D060829:Arthropod Proteins; D002413:Cations, Divalent; D066329:Protein Aggregates; D003300:Copper; D000255:Adenosine Triphosphate; D001120:Arginine; D011392:Proline; D001122:Arginine Kinase; D005998:Glycine