CHARACTERIZATION OF THE S-1 SUBSITE SPECIFICITY OF ASPERGILLOPEPSIN-IBY SITE-DIRECTED MUTAGENESIS

The structural determinants of Si substrate specificity of aspergillop epsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus s aitoi, were investigated by site-directed mutagenesis. Aspartic protei nases 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 asparti c proteinases, Asp-76 and Ser-78 of API are conserved only in fungal e nzymes with the ability to activate trypsinogen, and are located in th e active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78 A, and Delta S78) mere 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 drasti c decreases in the activities towards substrates containing a basic am ino acid residue at P-1. In contrast, substrates with a hydrophobic re sidue 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 re sidue at the P-1 position.