Advertisement

Substrate Specificity of Natural Variants and Genetically Engineered Intermediates of Bacillus Lentus Alkaline Proteases

  • Karl-Heinz Maurer
  • Martina Markgraf
  • Dean Goddette
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 379)

Summary

Three natural variants of subtilisin lentus could be differentiated by their amino acid sequence and their specific activity with low molecular weight peptide substrates of the type sAAPFpNA. The variants had amino acid exchanges in five, respective six positions of their amino acid sequence, four of which are located in the substrate loop of the enzyme (positions 92 – 102). Variants of one type of highly alkaline subtilisin (subtilisin 309) were made by site directed mutagenesis, each containing one of the corresponding amino acid exchanges. These intermediate forms were tested for activity, pH-dependence and substrate specificity. The changes in substrate affinity were relatively small for substrates with different amino acids as PI residue. The differences in activity on peptide-substrates could be related primarily to a single amino acid substitution in the S4 substrate binding pocket in position 102.

With subtrate variations in the P3 amino acid residue, changes in kcat and Km revealed the importance of the charged amino acid exchanged between subtilisin 309 and BLAP. By these experiments an interaction of amino acid position 101 and the P3 residue of the substrate could be demonstrated.

The substitution of two differently charged amino acids in the substrate binding region resulted in an unchanged pH-profile of the natural enzyme. With the single exchange intermediates differences in the pH-profile could be found, defending on the substrate tested: a characteristic change was observed with casein as substrate, no such change occurred with hemoglobin.

Keywords

Amino Acid Position Charge Amino Acid Peptide Substrate Amino Acid Exchange Substrate Binding Region 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    W.V. Shaw, Biochem. J.246: 1 (1987).PubMedGoogle Scholar
  2. 2.
    J.A. Wells and D.A. Estell, Trends Biochem. Sci.13: 291 (1988).PubMedCrossRefGoogle Scholar
  3. 3.
    I. Schechter and A. Berger, Biochem. Biophys. Res. Commun.27: 157 (1967).PubMedCrossRefGoogle Scholar
  4. 4.
    A.V. Teplyakov, J.M. van der Laan, A.A. Lammers, H. Kelder, K.H. Kalk, O. Misset, L.J.S.M. Mulleners, and B.W. Dijkstra, Protein Eng. 5: 413 (1992).PubMedCrossRefGoogle Scholar
  5. C.R. Wilson, B.F. Ladin, J.R. Mielenz, S.S.M. Horn, D. Hansen, R.B. Reynolds, N.C.R. Kennedy, J. Schindler, M. Bahn, R. Schmid, M. Markgraf, and C. Paech, PCT Patent Appl. WO 9102792, published March 7, 1991.Google Scholar
  6. S. Hastrup, S. Branner, F. Norris, S.B. Petersen, L. Norskov-Lauridsen, V.J. Jensen, and D. Aaslyng, PCT Patent Appl. WO 8906279, published July 13 1989.Google Scholar
  7. 7.
    J.C. van der Laan, G. Gerritse, L.J.S.M. Mulleners, R.A.C. van der Hoek, and W.J. Quax, Appl. Environ. Microbiol., 57: 901 (1991).PubMedGoogle Scholar
  8. 8.
    R.H. Doi, S. Wong, and F. Kawamura, Trends Biotechnol4: 232 (1986).CrossRefGoogle Scholar
  9. 9.
    P. Stanssens, C. Opsomer, Y. McKeown, W. Kramer, M. Zabeau, and H.J. Fritz, Nucleic Acids Res. 16: 6987 (1989).Google Scholar
  10. 10.
    R. Schülein, J. Kreft, S. Gonski, and W. Goebel, Mol. Gen. Genet.227: 137 (1991).PubMedCrossRefGoogle Scholar
  11. 11.
    E.G. Del Mar, C. Largman, J.W. Brodrick, and M.C. Geokas, Anal. Biochem.99: 316 (1979).CrossRefGoogle Scholar
  12. 12.
    P.E. Butler, M.J. McKay, and J.S. Bond, Biochem. J.241: 229 (1987).PubMedGoogle Scholar
  13. 13.
    J. Bates, J. Res. Natl Bur. Stand.66A: 179 (1962).Google Scholar
  14. 14.
    T. Maniatis, E. Fritsch, and J. Sambrook, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbour Laboratory, Cold Spring Harbour, N. Y. (1982).Google Scholar
  15. 15.
    R.E. Brown, K.L. Jarvis, and K.J. Hyland, Anal. Biochem.180: 136 (1989).PubMedCrossRefGoogle Scholar
  16. 16.
    R.J. Siezen, W.M. de Vos, J.A.M. Leunissen, and B.W. Dijkstra, Protein Eng. 4: 719 (1991).PubMedCrossRefGoogle Scholar
  17. A.J. Russell, P.G. Thomas, and A.R. Fersht J. Mol. Biol. 193: 803 (1987).Google Scholar
  18. 18.
    J. Kraut, Annu. Rev. Biochem.46: 331 (1977).PubMedCrossRefGoogle Scholar
  19. 19.
    W. Bode, E. Papamokos, and D. Musil, Eur. J. Biochem.166: 673 (1987).PubMedCrossRefGoogle Scholar
  20. 20.
    J.A. Wells, D.B. Powers, R.C. Bott, T.P. Graycar, and D.A. Estell, Proc. Natl. Acad. Sci. USA84: 1219 (1987).PubMedCrossRefGoogle Scholar
  21. 21.
    P.G. Thomas, A.J. Russell, and A.R. Fersht, Nature(London) 318: 375 (1985).Google Scholar
  22. 22.
    A.J. Russell and A.R. Fersht, Nature(London) 328: 496 (1987).Google Scholar
  23. 23.
    D.W. Goddette, C. Paech, S.S. Yang, J.R. Mielenz, C. Bystroff, M. Wilke, and R.J. Fletterick, J. Mol. Biol.228: 580 (1992).Google Scholar
  24. 24.
    R. Kaneko, N. Koyama, Y.-C. Tsai, R.-Y. Juang, K. Yoda, andM. Yamasaki, J. Bacteriol. 171: 5232 (1989).Google Scholar

Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • Karl-Heinz Maurer
    • 1
  • Martina Markgraf
    • 2
  • Dean Goddette
    • 3
  1. 1.COGNIS GmbHDüsseldorfGermany
  2. 2.Henkel Hygiene GmbHDüsseldorfGermany
  3. 3.COGNIS, IncSanta RosaUSA

Personalised recommendations