2008年3月
Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
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- 巻
- 1784
- 号
- 3
- 開始ページ
- 481
- 終了ページ
- 488
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.bbapap.2007.12.004
- 出版者・発行元
- ELSEVIER SCIENCE BV
Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, the k(cat)/K-m values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl2 at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl2 and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca2+-binding structures. (C) 2007 Elsevier B.V. All rights reserved.
- リンク情報
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- DOI
- https://doi.org/10.1016/j.bbapap.2007.12.004
- J-GLOBAL
- https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902281974080533
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/18187054
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000254780200006&DestApp=WOS_CPL
- ID情報
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- DOI : 10.1016/j.bbapap.2007.12.004
- ISSN : 1570-9639
- eISSN : 0006-3002
- J-Global ID : 200902281974080533
- PubMed ID : 18187054
- Web of Science ID : WOS:000254780200006