Characterization of the S 1 subsite specificity of aspergillopepsin I by site-directed mutagenesis

Takahiro Shintani, Mizue Kobayashi, Eiji Ichishima

    Research output: Contribution to journalArticlepeer-review

    18 Citations (Scopus)


    The structural determinants of S 1 substrate specificity of aspergillopepsin I (API; EC, an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P 1 and P 1'. However, API accommodates a Lys residue at P 1, which leads to activation of trypsinogen. On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and ΔS78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates. Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P 1. In contrast, substrates with a hydrophobic residue at P 1 were effectively hydrolyzed by each mutant enzyme. These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P 1 position.

    Original languageEnglish
    Pages (from-to)974-981
    Number of pages8
    JournalJournal of biochemistry
    Issue number5
    Publication statusPublished - 1996 Dec 10


    • Aspartic proteinase
    • Aspergillopepsin I
    • Site-directed mutagenesis
    • Substrate specificity
    • Trypsinogen activation

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology


    Dive into the research topics of 'Characterization of the S 1 subsite specificity of aspergillopepsin I by site-directed mutagenesis'. Together they form a unique fingerprint.

    Cite this