ENGINEERING OF PORCINE PEPSIN - ALTERATION OF S-1 SUBSTRATE-SPECIFICITY OF PEPSIN TO THOSE OF FUNGAL ASPARTIC PROTEINASES BY SITE-DIRECTED MUTAGENESIS

The S-1 substrate specificity of porcine pepsin has been altered to re semble that of fungal aspartic proteinase with preference for a basic amino acid residue in P-1 by site directed mutagenesis. On the basis o f primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved t he replacement of Thr-77 by Asp (T77D), the insertion of Ser between G ly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79) . The specificities of the mutants were determined using p-nitrophenyl alanine-based substrates containing a Phe or Lys residue at the P-1 po sition. The double mutant cleaved the Lys-Phe(4-NO2) bonds, while wild -type enzyme digested other bonds. In addition, the pH dependence of h ydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P-1 Lys contrib ute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleava ge of the Lys(6)-Ile(7) bond of trypsinogen. Results of this study sug gest that the structure of the active site flap contributes to the S-1 substrate specificity for basic amino acid residues in aspartic prote inases.