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. 2000 Oct;9(10):1947–1959. doi: 10.1110/ps.9.10.1947

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

C A Galea 1, B P Dalrymple 1, R Kuypers 1, R Blakeley 1
PMCID: PMC2144476  PMID: 11106168

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 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.

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Selected References

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  1. Albert A., Blundell T. L., Dhanaraj V., Donate L. E., Groves M., Guruprasad K., Nugent P. G., Orprayoon P., Pitts J. E., Rufino S. Protein engineering aspartic proteinases. Site-directed mutagenesis, biochemical characterisation, and X-ray analysis of chymosins with substituted single amino acid substitutions and loop replacements. Adv Exp Med Biol. 1998;436:169–177. [PubMed] [Google Scholar]
  2. Asghar A., Henrickson R. L. Chemical, biochemical, functional, and nutritional characteristics of collagen in food systems. Adv Food Res. 1982;28:231–372. doi: 10.1016/s0065-2628(08)60113-5. [DOI] [PubMed] [Google Scholar]
  3. Baldwin E. T., Bhat T. N., Gulnik S., Hosur M. V., Sowder R. C., 2nd, Cachau R. E., Collins J., Silva A. M., Erickson J. W. Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6796–6800. doi: 10.1073/pnas.90.14.6796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beyer B. M., Dunn B. M. Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, "short" pseudocathepsin D. J Biol Chem. 1996 Jun 28;271(26):15590–15596. doi: 10.1074/jbc.271.26.15590. [DOI] [PubMed] [Google Scholar]
  5. Chen L., Erickson J. W., Rydel T. J., Park C. H., Neidhart D., Luly J., Abad-Zapatero C. Structure of a pepsin/renin inhibitor complex reveals a novel crystal packing induced by minor chemical alterations in the inhibitor. Acta Crystallogr B. 1992 Aug 1;48(Pt 4):476–488. doi: 10.1107/s0108768192001939. [DOI] [PubMed] [Google Scholar]
  6. Conner G. E., Richo G. Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D. Biochemistry. 1992 Feb 4;31(4):1142–1147. doi: 10.1021/bi00119a024. [DOI] [PubMed] [Google Scholar]
  7. Dauber-Osguthorpe P., Roberts V. A., Osguthorpe D. J., Wolff J., Genest M., Hagler A. T. Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins. 1988;4(1):31–47. doi: 10.1002/prot.340040106. [DOI] [PubMed] [Google Scholar]
  8. Dealwis C. G., Frazao C., Badasso M., Cooper J. B., Tickle I. J., Driessen H., Blundell T. L., Murakami K., Miyazaki H., Sueiras-Diaz J. X-ray analysis at 2.0 A resolution of mouse submaxillary renin complexed with a decapeptide inhibitor CH-66, based on the 4-16 fragment of rat angiotensinogen. J Mol Biol. 1994 Feb 11;236(1):342–360. doi: 10.1006/jmbi.1994.1139. [DOI] [PubMed] [Google Scholar]
  9. Dunn B. M., Jimenez M., Parten B. F., Valler M. J., Rolph C. E., Kay J. A systematic series of synthetic chromophoric substrates for aspartic proteinases. Biochem J. 1986 Aug 1;237(3):899–906. doi: 10.1042/bj2370899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunn B. M., Scarborough P. E., Davenport R., Swietnicki W. Analysis of proteinase specificity by studies of peptide substrates. The use of UV and fluorescence spectroscopy to quantitate rates of enzymatic cleavage. Methods Mol Biol. 1994;36:225–243. doi: 10.1385/0-89603-274-4:225. [DOI] [PubMed] [Google Scholar]
  11. Fernley H. N. Statistical estimations in enzyme kinetics. The integrated Michaelis equation. Eur J Biochem. 1974 Apr 1;43(2):377–378. doi: 10.1111/j.1432-1033.1974.tb03423.x. [DOI] [PubMed] [Google Scholar]
  12. Fruton J. S. The mechanism of the catalytic action of pepsin and related acid proteinases. Adv Enzymol Relat Areas Mol Biol. 1976;44:1–36. doi: 10.1002/9780470122891.ch1. [DOI] [PubMed] [Google Scholar]
  13. Fujinaga M., Chernaia M. M., Tarasova N. I., Mosimann S. C., James M. N. Crystal structure of human pepsin and its complex with pepstatin. Protein Sci. 1995 May;4(5):960–972. doi: 10.1002/pro.5560040516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilliland G. L., Oliva M. T., Dill J. Functional implications of the three-dimensional structure of bovine chymosin. Adv Exp Med Biol. 1991;306:23–37. doi: 10.1007/978-1-4684-6012-4_3. [DOI] [PubMed] [Google Scholar]
  15. Gilliland G. L., Winborne E. L., Nachman J., Wlodawer A. The three-dimensional structure of recombinant bovine chymosin at 2.3 A resolution. Proteins. 1990;8(1):82–101. doi: 10.1002/prot.340080110. [DOI] [PubMed] [Google Scholar]
  16. Jupp R. A., Dunn B. M., Jacobs J. W., Vlasuk G., Arcuri K. E., Veber D. F., Perlow D. S., Payne L. S., Boger J., de Laszlo S. The selectivity of statine-based inhibitors against various human aspartic proteinases. Biochem J. 1990 Feb 1;265(3):871–878. doi: 10.1042/bj2650871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Khan A. R., James M. N. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci. 1998 Apr;7(4):815–836. doi: 10.1002/pro.5560070401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Knight C. G. Active-site titration of peptidases. Methods Enzymol. 1995;248:85–101. doi: 10.1016/0076-6879(95)48008-0. [DOI] [PubMed] [Google Scholar]
  19. Kuypers R., Tyler M., Kurth L. B., Jenkins I. D., Horgan D. J. Identification of the loci of the collagen-associated Ehrlich chromogen in type I collagen confirms its role as a trivalent cross-link. Biochem J. 1992 Apr 1;283(Pt 1):129–136. doi: 10.1042/bj2830129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin X. L., Lin Y. Z., Tang J. Relationships of human immunodeficiency virus protease with eukaryotic aspartic proteases. Methods Enzymol. 1994;241:195–224. doi: 10.1016/0076-6879(94)41066-6. [DOI] [PubMed] [Google Scholar]
  21. Lin Y., Fusek M., Lin X., Hartsuck J. A., Kezdy F. J., Tang J. pH dependence of kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants. J Biol Chem. 1992 Sep 15;267(26):18413–18418. [PubMed] [Google Scholar]
  22. Marciniszyn J., Jr, Hartsuck J. A., Tang J. Mode of inhibition of acid proteases by pepstatin. J Biol Chem. 1976 Nov 25;251(22):7088–7094. [PubMed] [Google Scholar]
  23. Mechanic G. L., Katz E. P., Henmi M., Noyes C., Yamauchi M. Locus of a histidine-based, stable trifunctional, helix to helix collagen cross-link: stereospecific collagen structure of type I skin fibrils. Biochemistry. 1987 Jun 16;26(12):3500–3509. doi: 10.1021/bi00386a038. [DOI] [PubMed] [Google Scholar]
  24. Medzihradzky K., Voynick I. M., Medzihradszky-Schweiger H., Fruton J. S. Effect of secondary enzyme-substrate interactions on the cleavage of synthetic peptides by pepsin. Biochemistry. 1970 Mar 3;9(5):1154–1162. doi: 10.1021/bi00807a016. [DOI] [PubMed] [Google Scholar]
  25. Newman M., Safro M., Frazao C., Khan G., Zdanov A., Tickle I. J., Blundell T. L., Andreeva N. X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin. J Mol Biol. 1991 Oct 20;221(4):1295–1309. [PubMed] [Google Scholar]
  26. Nimmo I. A., Atkins G. L. A comparison of two methods for fitting the integrated Michaelis-Menten equation. Biochem J. 1974 Sep;141(3):913–914. doi: 10.1042/bj1410913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rao C., Dunn B. M. Evidence for electrostatic interactions in the S2 subsite of porcine pepsin. Adv Exp Med Biol. 1995;362:91–94. doi: 10.1007/978-1-4615-1871-6_10. [DOI] [PubMed] [Google Scholar]
  28. Scarborough P. E., Dunn B. M. Redesign of the substrate specificity of human cathepsin D: the dominant role of position 287 in the S2 subsite. Protein Eng. 1994 Apr;7(4):495–502. doi: 10.1093/protein/7.4.495. [DOI] [PubMed] [Google Scholar]
  29. Scarborough P. E., Guruprasad K., Topham C., Richo G. R., Conner G. E., Blundell T. L., Dunn B. M. Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling. Protein Sci. 1993 Feb;2(2):264–276. doi: 10.1002/pro.5560020215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  31. Scott P. G., Pearson H. Cathepsin D: specificity of peptide-bond cleavage in type-I collagen and effects on type-III collagen and procollagen. Eur J Biochem. 1981;114(1):59–62. doi: 10.1111/j.1432-1033.1981.tb06172.x. [DOI] [PubMed] [Google Scholar]
  32. Yamauchi M., London R. E., Guenat C., Hashimoto F., Mechanic G. L. Structure and formation of a stable histidine-based trifunctional cross-link in skin collagen. J Biol Chem. 1987 Aug 25;262(24):11428–11434. [PubMed] [Google Scholar]
  33. al-Janabi J., Hartsuck J. A., Tang J. Kinetics and mechanism of pepsinogen activation. J Biol Chem. 1972 Jul 25;247(14):4628–4632. [PubMed] [Google Scholar]

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