Gly or Ala substitutions for Pro²¹⁰Thr²¹¹Asn²¹² at the 尾8-尾9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability

详细信息	查看全文 | 推荐本文 |

• 作者：Naoki Fuchita^a ; ^b ; Saori Arita^a ; Junya Ikuta^a ; ^b ; Masahiro Miura^a ; Kaori Shimomura^a ; Hiroyuki Motoshima^a ; Keiichi Watanabe^a ; ^b ; ^{watakei@cc.saga-u.ac.jp}
• 关键词：sC ; subtilisin Carlsberg ; AAPF ; N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide ; AAPL ; N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide ; AAVA ; N-succinyl-Ala-Ala-Val-Ala-p-nitroanilide ; PTN ; wild-type ; GTN ; P210G mutant ; PGN ; T211G mutant ; PTG ; N212G mutant ; ATN ; P210
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2012
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：April, 2012
• 卷：1824
• 期：4
• 页码：620-626
• 文件大小：566 K

摘要

A comparison of the primary structures among psychrophilic, mesophilic, and thermophilic subtilases revealed that the turn between the 尾8 and 尾9 strands (尾8-尾9 turn, BPN鈥?numbering) of psychrophilic subtilases are more flexible than those of their mesophilic and thermophilic counterparts. To investigate the relationship between structure of this turn and enzyme activity as well as thermostability of mesophilic subtilisin Carlsberg (sC), we analyzed 6 mutants of sC with a single, double, or triple Gly or Ala substitutions for Pro²¹⁰Thr²¹¹Asn²¹² at the 尾8-尾9 turn. Among the single Gly substitutions, the P210G substitution most significantly (1.5-fold) increased the specific activity on N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF) substrate and 12-fold decreased the thermostability. All mutants tested showed the increased k_cat for the AAPF substrate and reduced thermostability compared with the wild-type sC. The k_cat values of the P210G, P210G/T211G, and P210G/T211G/N212G mutants were 1.5-, 1.7-, and 1.8-fold higher than that of the wild-type sC. There were significant positive correlations between k_cat and thermal inactivation rates as well as k_cat and K_m of the wild-type and mutants. These results demonstrate that the structure of 尾8-尾9 turn, despite its distance from the active site, has significant effects on the catalytic rate and thermostability of sC through a global network of intramolecular interactions and suggest that the lack of flexibility of this turn stabilizes the wild-type sC against thermal inactivation in compensation for some loss of catalytic activity.