Protein Science, Vol 9, Issue 10 1947-1959, Copyright © 2000 by The Protein Society

Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin [In Process Citation]

CA Galea, BP Dalrymple, R Kuypers and R Blakeley
Department of Biochemistry, University of Queensland, St. Lucia, Australia.

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 alpha1 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.
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?