The S 1 substrate specificity of porcine pepsin has been altered to resemble that of fungal aspartic proteinase with preference for a basic amino acid residue in P 1 by site directed mutagenesis. On the basis of primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved the replacement of Thr-77 by Asp (T77D), the insertion of Ser between Gly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79). The specificities of the mutants were determined using p-nitrophenylalanine-based substrates containing a Phe or Lys residue at the P 1 position. The double mutant cleaved the Lys-Phe(4-NO 2 ) bonds, while wild-type enzyme digested other bonds. In addition, the pH dependence of hydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P 1 Lys contribute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys 6 -Ile 7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contributes to the S 1 substrate specificity for basic amino acid residues in aspartic proteinases.Aspartic endopeptidases (EC 3.4.23._) comprise a group of enzymes whose proteolytic activities are dependent on two aspartyl residues, Asp-32 and Asp-215, in pepsin numbering (1). Mammalian and fungal enzymes have been extensively characterized, and their three-dimensional structure has been determined at high resolution (2-7). The enzymes of this family are bilobal with a deep and extended cleft which can accommodate at least seven amino acid residues in the S 4 -S 3 Ј subsites 1 (8). The "flap," an antiparallel ␤-hairpin loop comprising residues 72 to 82 (pepsin numbering), projects across the cleft forming a channel into which a substrate binds. Although the enzymes are quite similar in their three-dimensional structures, there are drastic differences in the catalytic properties, especially in substrate specificities. There also have been many studies on the tertiary structures of aspartic proteinases with peptide-derived inhibitors (4, 5, 9 -14), and the relationship between their structures and substrate specificities has been demonstrated (4, 5, 9 -13, 15, 16). According to these reports, although the hydrogen bonding pattern between the main chain of inhibitor and enzyme is well conserved in all inhibitor-enzyme complexes, the differences in the size of subsites, of which residues make van der Waals contacts with the side chain of inhibitor, may control the substrate specificities in aspartic proteinases. Several attempts to examine the structural determinants of substrate specificities of aspartic proteinases by site-directed mutagenesis have been made recently (17-23). These provided direct evidence that the subtle differences in the structures of substrate binding sites of aspartic proteinases were sufficient to alter their substrate specificities.Aspartic pr...