Gly or Ala substitutions for Pro210Thr211Asn212 at the β8–β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability
Autor: | Junya Ikuta, Kaori Shimomura, Hiroyuki Motoshima, Masahiro Miura, Keiichi Watanabe, Saori Arita, Naoki Fuchita |
---|---|
Rok vydání: | 2012 |
Předmět: |
Threonine
Proline Stereochemistry Amino Acid Motifs Molecular Sequence Data Glycine Biophysics Bacillus Biochemistry Subtilase Analytical Chemistry Turn (biochemistry) Bacterial Proteins Catalytic Domain Enzyme Stability Amino Acid Sequence Subtilisins Enzyme kinetics Psychrophile Molecular Biology Thermostability Alanine Sequence Homology Amino Acid biology Chemistry Subtilisin Active site Substrate (chemistry) Kinetics Crystallography Amino Acid Substitution Proteolysis Biocatalysis Mutagenesis Site-Directed biology.protein Asparagine Peptides |
Zdroj: | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1824:620-626 |
ISSN: | 1570-9639 |
DOI: | 10.1016/j.bbapap.2012.01.015 |
Popis: | A comparison of the primary structures among psychrophilic, mesophilic, and thermophilic subtilases revealed that the turn between the β8 and β9 strands (β8-β9 turn, BPN' 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 Pro(210)Thr(211)Asn(212) at the β8-β9 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 β8-β9 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. |
Databáze: | OpenAIRE |
Externí odkaz: |