Engineering of Porcine Pepsin

Abstract

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...

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

“…Since cleavage at the mutated ϳVal 29 -Phe 30 ϳ bond was no longer an option, it was found by amino acid analysis that processing had taken place at the adjacent ϳPhe 30 *Lys 31 ϳ bond (data not shown). Similarly, peptides 12 and 13 (Table II) were digested after 72 h incubation with proteinase A at a molar ratio of 10:1, as were peptides 18,19,20, and 26 of the peptides listed in Table IV. Thus, in addition to the L19A mutant (peptide 27) as described above, the other peptides (7,8,12,13,14 (Table II), 18, 19, 20, and 26 (Table IV)) which were not effective as inhibitors of proteinase A, served instead as substrates for the enzyme.…”

The Evolution of Pepsinogen C Genes in Vertebrates: Duplication, Loss and Functional Diversification

“…1 green boxes) bind to the catalytic aspartates (Fig. 1 black boxes “+”) [1], [26], [27], [28] In an acidic pH environment acidic residues in the enzyme moiety become protonated disrupting electrostatic interactions with the prosegment (which has a basic character), releasing the prosegment for proteolytic cleavage and enzyme activation [3], [29], [30]. In fish pepsinogens a deletion of several residues in the prosegment is observed (Fig.…”

Structure of aspergillopepsin I fromAspergillus phoenicis: variations of the S1′–S2 subsite in aspartic proteinases

“…In all mammalian proteins that favour a hydrophobic P1 residue, Asp76 is substituted by Thr or Ser and Ser78 is deleted. It has been suggested that Asp76 interacts with P1 lysine, contributing to the transition-state stabilization, and that Ser78 is important for the proper orientation of the side chain of Asp76 (Shintani et al, 1997). The hydrogen bond between Asp76 and Ser78 is indeed present in molecule A but not in B.…”

“…Prediction of the binding preferences from the amino-acid sequence is dif®cult because the speci®city is determined by extensive interactions occurring at multiple subsites and the shape of each subsite is delineated by the structures of the¯exible loops as well as the individual aminoacid residues in the loops (Dunn & Hung, 2000). Site-directed mutagenesis showed that a single mutation at the binding site alters the substrate speci®city (Scarborough & Dunn, 1994;Shintani et al, 1997).…”