2017年8月
Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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- 巻
- 101
- 号
- 開始ページ
- 653
- 終了ページ
- 659
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.ijbiomac.2017.03.133
- 出版者・発行元
- ELSEVIER SCIENCE BV
The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant.
In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G29812/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.(C) 2017 Elsevier B.V. All rights reserved.
In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G29812/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.(C) 2017 Elsevier B.V. All rights reserved.
- リンク情報
- ID情報
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- DOI : 10.1016/j.ijbiomac.2017.03.133
- ISSN : 0141-8130
- eISSN : 1879-0003
- Web of Science ID : WOS:000404491300072