Restriction of substrate specificity of subtilisin E by introduction of a side chain into a conserved glycine residue

FEBS Letters

Takagi, Hiroshi²  Tsai, Yeng-Chieh¹  Nakamori, Shigeru²  Maeda, Tadao²  Ohtsu, Iwao² 

[1] Institute of Biochemistry, National Yang-Ming Medical College, Shih-Pai, Taipei, 11221, Taiwan, ROC;Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Yoshida-gun, Fukui 910-11, Japan

关键词: Subtilisin;    Substrate specificity;    Site-directed mutagenesis;   

DOI  :  10.1016/0014-5793(96)01014-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Substitution of the conserved Gly¹²⁷ for residues having a side chain markedly changed the substrate specificity of subtilisin E from Bacillus subtilis. The crystallographic findings suggested that Gly¹²⁷ is responsible for accepting even the large P1 substrates, and the marked change of specificity was attributed to the introduction of a side chain in this position. To test this hypothesis, Gly¹²⁷ was replaced with 3 non-charged amino acids, Ala, Ser and Val. When assayed with synthetic peptide substrates, all mutants purified from the periplasmic space in Escherichia coli showed a marked preference for small P1 substrate up to 150-fold relative to the wild-type. The kinetic data and molecular modeling analysis suggest that large hydrophobic P1 residues were unable to access the binding pocket due to steric hindrance.

【 授权许可】

Unknown