Gly or Ala substitutions for Pro(210)Thr(211)Asn(212) at the β8-β9 turn of subtilisin Carlsberg increase the catalytic rate and decrease thermostability.
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.
Department of Applied Biochemistry and Food Science, Saga University, Saga, Japan.
Naoki
N
Fuchita
Saori
S
Arita
Junya
J
Ikuta
Masahiro
M
Miura
Kaori
K
Shimomura
Hiroyuki
H
Motoshima
Keiichi
K
Watanabe
D000409:Alanine / Q000737:chemistry / Q000235:genetics*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D004795:Enzyme Stability; D005998:Glycine / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry / Q000235:genetics; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics; D013912:Threonine / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D010455:Peptides; D013912:Threonine; D001216:Asparagine; D011392:Proline; D013381:Subtilisins; D000409:Alanine; D005998:Glycine