Gly or Ala substitutions for Pro210Thr211Asn212 at the β8–β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability

• Published in: Biochimica et biophysica acta. Proteins and proteomics Vol. 1824; no. 4; pp. 620 - 626
• Main Authors: Fuchita, Naoki, Arita, Saori, Ikuta, Junya, Miura, Masahiro, Shimomura, Kaori, Motoshima, Hiroyuki, Watanabe, Keiichi
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.04.2012
• Subjects: active sites; catalytic activity; Catalytic rate; correlation; enzyme activity; Flexibility; heat inactivation; mutants; Proline; Subtilisin; thermal stability; Thermostability; β turn; GTN; Catalytic rate; AAPL; PTG; GGG; AAPF; Proline; Subtilisin; β turn; PTN; Flexibility; GGN; sC; Thermostability; AAVA; PGN; ATN
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2012.01.015

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 Pro210Thr211Asn212 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 kcat for the AAPF substrate and reduced thermostability compared with the wild-type sC. The kcat 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 kcat and thermal inactivation rates as well as kcat and Km 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. ► We substituted Gly or Ala for Pro-Thr-Asn at β8–β9 turn of subtilisin Carlsberg (sC). ► This turn is located apart from the active site. ► These mutations increased kcat for peptide hydrolysis and decreased thermostability. ► P210G mutation had the largest effects among single Gly substitutions for PTN residues. ► Positive correlations were found between kcat and thermal inactivation rates.