1999年1月
Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues
JOURNAL OF BIOLOGICAL CHEMISTRY
- ,
- ,
- ,
- 巻
- 274
- 号
- 4
- 開始ページ
- 2344
- 終了ページ
- 2349
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1074/jbc.274.4.2344
- 出版者・発行元
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Directed evolution was used to change the substrate specificity of aspartate aminotransferase, A mutant enzyme with 17 amino acid substitutions was generated that; shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (k(cat)/K-m) for a non-native substrate, valine, The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-Angstrom resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding, The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.
- リンク情報
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- DOI
- https://doi.org/10.1074/jbc.274.4.2344
- CiNii Articles
- http://ci.nii.ac.jp/naid/80010895948
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/9891001
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000078185200068&DestApp=WOS_CPL
- ID情報
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- DOI : 10.1074/jbc.274.4.2344
- ISSN : 0021-9258
- CiNii Articles ID : 80010895948
- PubMed ID : 9891001
- Web of Science ID : WOS:000078185200068