title: Characterization of the S1 subsite specificity of aspergillopepsin I by site-directed mutagenesis.
abstract: The structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1'. However, API accommodates a Lys residue at P1, 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 delta S78) 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 P1. In contrast, substrates with a hydrophobic residue at P1 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 P1 position.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai.
authors.forename: T
authors.initials: T
authors.lastname: Shintani
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
authors.forename: E
authors.initials: E
authors.lastname: Ichishima
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C029094:aspergillopepsin I