Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin

Galea, Charles A., Dalrymple, Brian P., Kuypers, Ron and Blakeley, Robert (2000) Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin. Protein Science, 9 10: 1947-1959. doi:10.1110/ps.9.10.1947

Author Galea, Charles A.
Dalrymple, Brian P.
Kuypers, Ron
Blakeley, Robert
Title Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin
Journal name Protein Science   Check publisher's open access policy
ISSN 0961-8368
Publication date 2000-01-01
Sub-type Article (original research)
DOI 10.1110/ps.9.10.1947
Open Access Status DOI
Volume 9
Issue 10
Start page 1947
End page 1959
Total pages 13
Place of publication Hoboken, N. J .
Publisher Wiley-Blackwell Publishing
Language eng
Subject C1
270108 Enzymes
780105 Biological sciences
Formatted abstract
 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.
Keyword Biochemistry & Molecular Biology
Aspartic Protease
Cathepsin D
Chromogenic Substrate
Substrate Specificity
Human Cathepsin-d
Aspartic Proteinases
Systematic Series
S-2 Subsite
I Collagen
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 15 times in Thomson Reuters Web of Science Article | Citations
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Created: Tue, 10 Jun 2008, 21:24:33 EST