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. 1995 Nov;61(11):3934–3939. doi: 10.1128/aem.61.11.3934-3939.1995

Involvement of enzyme-substrate charge interactions in the caseinolytic specificity of lactococcal cell envelope-associated proteinases.

J R Reid 1, T Coolbear 1, C H Moore 1, D R Harding 1, G G Pritchard 1
PMCID: PMC167699  PMID: 8526506

Abstract

Three series of oligopeptides were synthesized to investigate the proposal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against caseins is the interactions between charged amino acids in the substrate and in the enzyme. The sequences of the oligopeptides were based on two regions of kappa-casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to PI- and PIII-type lactococcal proteinases. In each series, one oligopeptide had an identical sequence to that of the kappa-casein region, while in the others, one or more charged residues were substituted by an amino acid of opposite charge, i.e., His<-->Glu. Generally, substitution of His by Glu in the oligopeptides corresponding to residues 98 to 111 of kappa-casein resulted in reduced cleavage of susceptible bonds by the PI-type proteinase and increased cleavage of susceptible bonds by the PIII-type proteinase. In the case of the oligopeptide corresponding to residues 153 to 169 of kappa-casein, one major cleavage site was evident, and the bond was hydrolyzed by both types of proteinase (even though this sequence in kappa-casein itself is extremely resistant to the PI-type enzyme). Substitution of Glu by His in this oligopeptide, even in the P7 position, resulted in increased cleavage of the bond by the PI-type proteinase and reduced cleavage by the PIII-type proteinase. C-terminal truncation of this oligopeptide resulted in a 100-fold decrease in the rate of hydrolysis of the susceptible bond and a change in the pattern of cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Coolbear T., Reid J. R., Pritchard G. G. Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions. Appl Environ Microbiol. 1992 Oct;58(10):3263–3270. doi: 10.1128/aem.58.10.3263-3270.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Englebretsen D. R., Harding D. R. Solid phase peptide synthesis on hydrophilic supports. Part II--Studies using Perloza beaded cellulose. Int J Pept Protein Res. 1992 Dec;40(6):487–496. [PubMed] [Google Scholar]
  3. Exterkate F. A., Alting A. C., Bruinenberg P. G. Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region. Appl Environ Microbiol. 1993 Nov;59(11):3640–3647. doi: 10.1128/aem.59.11.3640-3647.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Exterkate F. A., Alting A. C., Slangen C. J. Specificity of two genetically related cell-envelope proteinases of Lactococcus lactis subsp. cremoris towards alpha s1-casein-(1-23)-fragment. Biochem J. 1991 Jan 1;273(Pt 1):135–139. doi: 10.1042/bj2730135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Exterkate F. A. Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp. cremoris are related to secondary binding specificity. Appl Microbiol Biotechnol. 1990 Jul;33(4):401–406. doi: 10.1007/BF00176654. [DOI] [PubMed] [Google Scholar]
  6. Kok J., Leenhouts K. J., Haandrikman A. J., Ledeboer A. M., Venema G. Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl Environ Microbiol. 1988 Jan;54(1):231–238. doi: 10.1128/aem.54.1.231-238.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Pritchard G. G., Coolbear T. The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol Rev. 1993 Sep;12(1-3):179–206. doi: 10.1111/j.1574-6976.1993.tb00018.x. [DOI] [PubMed] [Google Scholar]
  8. Raap J., Kerling K. E., Vreeman H. J., Visser S. Peptide substrates for chymosin (rennin): conformational studies of kappa-casein and some kappa-casein-related oligopeptides by circular dichroism and secondary structure prediction. Arch Biochem Biophys. 1983 Feb 15;221(1):117–124. doi: 10.1016/0003-9861(83)90127-3. [DOI] [PubMed] [Google Scholar]
  9. Reid J. R., Coolbear T., Pillidge C. J., Pritchard G. G. Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains. Appl Environ Microbiol. 1994 Mar;60(3):801–806. doi: 10.1128/aem.60.3.801-806.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Reid J. R., Moore C. H., Midwinter G. G., Pritchard G. G. Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine alpha s1-casein. Appl Microbiol Biotechnol. 1991 May;35(2):222–227. doi: 10.1007/BF00184690. [DOI] [PubMed] [Google Scholar]
  11. Reid J. R., Ng K. H., Moore C. H., Coolbear T., Pritchard G. G. Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp. cremoris [corrected]. Appl Microbiol Biotechnol. 1991 Dec;36(3):344–351. doi: 10.1007/BF00208154. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Siezen R. J., Bruinenberg P. G., Vos P., van Alen-Boerrigter I., Nijhuis M., Alting A. C., Exterkate F. A., de Vos W. M. Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis. Protein Eng. 1993 Nov;6(8):927–937. doi: 10.1093/protein/6.8.927. [DOI] [PubMed] [Google Scholar]
  14. Siezen R. J., de Vos W. M., Leunissen J. A., Dijkstra B. W. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Eng. 1991 Oct;4(7):719–737. doi: 10.1093/protein/4.7.719. [DOI] [PubMed] [Google Scholar]
  15. Tan P. S., Poolman B., Konings W. N. Proteolytic enzymes of Lactococcus lactis. J Dairy Res. 1993 May;60(2):269–286. doi: 10.1017/s0022029900027606. [DOI] [PubMed] [Google Scholar]
  16. Visser S., Exterkate F. A., Slangen C. J., de Veer G. J. Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine alpha(s1)-, beta-, and kappa-Casein. Appl Environ Microbiol. 1986 Nov;52(5):1162–1166. doi: 10.1128/aem.52.5.1162-1166.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Visser S., Slangen C. J., Robben A. J., van Dongen W. D., Heerma W., Haverkamp J. Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine kappa-casein. Appl Microbiol Biotechnol. 1994 Aug;41(6):644–651. doi: 10.1007/BF00167279. [DOI] [PubMed] [Google Scholar]
  18. Vos P., Boerrigter I. J., Buist G., Haandrikman A. J., Nijhuis M., de Reuver M. B., Siezen R. J., Venema G., de Vos W. M., Kok J. Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes. Protein Eng. 1991 Apr;4(4):479–484. doi: 10.1093/protein/4.4.479. [DOI] [PubMed] [Google Scholar]
  19. Vos P., Simons G., Siezen R. J., de Vos W. M. Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase. J Biol Chem. 1989 Aug 15;264(23):13579–13585. [PubMed] [Google Scholar]

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