کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1988221 | 1540317 | 2008 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
cDNAs of the two-domain arginine kinase (AK) (contiguous dimer; denoted by 2D/WT) and its separated domains 1 and 2 (denoted by D1/WT and D2/WT) from the sea anemone Anthopleura japonicus, were cloned into the plasmid pMAL, and recombinant enzymes were expressed in E. coli as MBP fusion proteins. The kinetic parameters kcat, Ka and Kia, were determined for all three AKs. All three enzymes showed distinct AK activity, and had high affinity for arginine (KaArg=0.25-0.48mM). The catalytic efficiency, calculated by kcat/KaArgKiaATP, of the 2D/WT enzyme (182Â mMâ2Â sâ1, the value for one active 40Â kDa domain) was two- to three-times higher than values for either D1/WT or D2/WT (80.2 and 86.4Â mMâ2Â sâ1, respectively), suggesting the presence of domain-domain interactions (cooperativity) in the contiguous dimer. The Kia/Ka values of the three enzymes ranged from 0.88 to 1.32, indicating that there is no strong synergism in substrate binding, as seen in typical AKs. Asp62 and Arg193, which are conserved in most AKs and play a key role in stabilizing the substrate-bound structure, are also conserved in the two domains of Anthopleura AK. We replaced Asp62 in D2/WT with Glu or Gly. The catalytic efficiency and Kia/Ka for the D62E mutant were comparable to those of D2/WT, but catalytic efficiency for the D62G mutant was decreased to 13% of that of the D2/WT with a significantly increased value of Kia/Ka (1.92), indicating that Asp62 plays an important role in the expression of AK activity.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: International Journal of Biological Macromolecules - Volume 42, Issue 1, 1 January 2008, Pages 46-51
Journal: International Journal of Biological Macromolecules - Volume 42, Issue 1, 1 January 2008, Pages 46-51
نویسندگان
Hiroshi Tada, Yuko Nishimura, Tomohiko Suzuki,