Kinetics for Cu(2+) induced Sepia pharaonis arginine kinase inactivation and aggregation.
Arginine kinase plays an important role in cellular energy metabolism and is closely related to the environmental stress response in marine invertebrates. We studied the Cu(2+)-mediated inhibition and aggregation of Sepia pharaonis arginine kinase (SPAK) and found that Cu(2+) markedly inhibited the SPAK activity along with mixed-type inhibition against the arginine substrate and noncompetitive inhibition against the ATP cofactor. Spectrofluorimetry results showed that Cu(2+) induced a tertiary structure change in SPAK, resulting in exposure of the hydrophobic surface and increased aggregation. Cu(2+)-mediated SPAK aggregation followed first-order kinetics consistent with monophasic and a biphasic processes. Addition of osmolytes, including glycine and proline, effectively blocked SPAK aggregation and restored SPAK activity. Our results demonstrated the effects of Cu(2+) on SPAK catalytic function, conformation, and aggregation, as well as the protective effects of osmolytes on SPAK folding. This study provided important insights into the role of Cu(2+) as a negative effector of the S. pharaonis metabolic enzyme AK and the possible responses of cephalopods to unfavorable environmental conditions.
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
Xiao-Yu
XY
Shi
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
Li-Li
LL
Zhang
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
Feng
F
Wu
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
Yang-Yong
YY
Fu
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
Shang-Jun
SJ
Yin
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China. Electronic address: siyuexiu@hotmail.com.
Yue-Xiu
YX
Si
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China; Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, PR China. Electronic address: parkyd@hotmail.com.
Yong-Doo
YD
Park
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001122:Arginine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D005998:Glycine / Q000494:pharmacology; D007700:Kinetics; D011392:Proline / Q000494:pharmacology; D066329:Protein Aggregates / Q000187:drug effects*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D049833:Sepia / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D066329:Protein Aggregates; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011392:Proline; D001122:Arginine Kinase; D005998:Glycine
Arginine kinase; Cu(2+); Inhibition