title: A folding study of Antarctic krill (Euphausia superba) alkaline phosphatase using denaturants.
abstract: To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH4)2SO4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography. The purified enzyme (62.6 kDa; 2.62 unit/mg) was unstable at temperatures exceeding 30°C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300 mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5 M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions.
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
authors.forename: Zhi-Jiang
authors.initials: ZJ
authors.lastname: Wang
authors.affiliation: Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea; Department of Bioinformatics, University of Sciences and Technology, Daejeon 305-350, Korea.
authors.forename: Jinhyuk
authors.initials: J
authors.lastname: Lee
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
authors.forename: Yue-Xiu
authors.initials: YX
authors.lastname: Si
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710, Korea.
authors.forename: Jun-Mo
authors.initials: JM
authors.lastname: Yang
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
authors.forename: Shang-Jun
authors.initials: SJ
authors.lastname: Yin
authors.affiliation: College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China. Electronic address: qianguoying_wanli@hotmail.com.
authors.forename: Guo-Ying
authors.initials: GY
authors.lastname: Qian
authors.affiliation: 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.
authors.forename: Yong-Doo
authors.initials: YD
authors.lastname: Park
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D033201:Euphausiacea / Q000201:enzymology*; D019791:Guanidine / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D012967:Sodium Dodecyl Sulfate; D014508:Urea; D000469:Alkaline Phosphatase; D019791:Guanidine
keywords: Alkaline phosphatase; Antarctic krill; Folding