The substrate specificity of porcine pepsin has been
altered by site-directed mutagenesis in an attempt to
selectively cleave bovine hide collagen at only a few
sites, similar to cathepsin D, for the production of high
quality gelatin. Kinetic parameters were determined using
chromogenic peptide substrates based on the sequence
Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro,
Yaa is Glu, Leu, Gln or Lys, Nph is p-nitrophenylalanine,
and * is the site of cleavage). Substitution of Thr222 and Glu287
within the S2 subsite of pepsin by Val and Met,
respectively, produced a double mutant with a two- to fourfold higher
kcat/Km,
compared with wild-type pepsin, for the chromogenic peptides with
residues Leu, Gln, and Glu at position P2 (Yaa).
The results suggest that the functional group of the P2
side chain may be exposed to solvent, while the aliphatic
portion interacts with hydrophobic residues comprising
S2. Wild-type pepsin cleaved a peptide corresponding
to the carboxy-terminal telopeptide region of bovine type
I collagen α1 chain, SGGYDLSFLPQPPQE, predominantly
at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a
significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met
cleaved site Ser-Phe at a rate 20-fold higher than the
wild-type. Significantly, enzymes containing the double
substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly
at one site Leu-Ser (similar to cathepsin D) and at a rate
23-fold higher than the wild-type. These mutants can potentially
enhance the rate of solubilization of bovine hide collagen
under conditions mild enough to maintain the triple helix
structure and hence minimize the rate of subsequent denaturation
and proteolytic cleavage.