Structure-Function Relationships in Lima Bean Protease Inhibitor

  • F. C. Stevens
  • S. Wuerz
  • J. Krahn
Part of the Bayer-Symposium book series (BAYER-SYMP, volume 5)


At the First International Research Conference on Proteinase Inhibitors we reported our findings on lima bean protease inhibitor (LBI)1 up to that time [1]. We confirmed previous observations [2, 3] that commercial LBI could be separated into four to six variants which are all biologically active and resemble each other strongly in amino acid composition. By isolation of the enzyme-inhibitor complexes by gel filtration and subsequent amino acid analysis we demonstrated that LBI could form 1:1 molar complexes with trypsin and also with chymotrypsin, that the binding sites for these two enzymes are different and independent and that one can also obtain an enzymatically inactive ternary complex containing one mole each of trypsin, chymotrypsin and LBI [4]. Using the partial proteolysis technique pioneered by Laskowski, Jr. et ai [5], we were able to show that LBI contains a particularly chymotrypsin sensitive bond, identified it as Leu55-Ser56 and suggested it was the chymotrypsin reactive site of LBI [6]; similar studies using trypsin indicated that there was a trypsin sensitive Lys-X bond in the inhibitor but at the time of the last conference we had not further identified or characterized this bond. We also presented a tentative amino acid sequence for one of the variants of LBI (variant IV) and noted that our starting material for the sequence determination was microheterogeneous and contained at least two genetic variants. We also drew attention to the existence of two homologous regions approximately 12 amino acid residues long; one region contains the chymotrypsin sensitive Leu55-Ser56 peptide bond and the corresponding residues in the other region are Lys28-Ser29 suggesting that this may be the trypsin reactive site and that this double headed inhibitor had arisen by partial gene duplication. The details of the amino acid sequence determination have been published [7, 8]. In further studies carried out since the last conference we have positively identified Lys28-Ser29 as the trypsin sensitive bond in LBI [9] and have also carried out an extensive study of the peptide bond hydrolysis equilibria governing this peptide bond, which confirm it as being the trypsin reactive site [10]. More recently we have directed our attention to the importance of the disulfide bonds for the biological activity of LBI [11] and to differences in biological activity between different naturally occurring variants of LBI [12].


Amino Acid Composition Reactive Site Peptide Bond Amino Acid Analysis Complete Homology 
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  1. 1.
    Stevens, F.C: In: Fritz, H., Tschesche, H. (Eds.): Proceedings of the International Research Conference on Proteinase Inhibitors, p. 149. Berlin-New York: Walter de Gruyter 1971.Google Scholar
  2. 2.
    Jones, G.. Moore, S.. Stein. W.H.: Biochemistry 2, 66 (1963).CrossRefGoogle Scholar
  3. 3.
    Haynes, R., Feeney, R.E.: J. biol. Chem. 242, 5378 (1967).PubMedGoogle Scholar
  4. 4.
    Krahn, J., Stevens, F.C: FEBS Letters 13, 339 (1971).PubMedCrossRefGoogle Scholar
  5. 5.
    Ozawa, K., Laskowski, M., Jr.: J. biol. Chem. 241, 3955 (1966).PubMedGoogle Scholar
  6. 6.
    Krahn, J., Stevens, F.C.: Biochemistry 9, 2646 (1970).PubMedCrossRefGoogle Scholar
  7. 7.
    Tan, C.G.L., Stevens, F.C.: Europ. J. Biochem. 18, 503 (1971).PubMedCrossRefGoogle Scholar
  8. 8.
    Tan, C.G.L., Stevens, F.C.: Europ. J. Biochem. 18, 515 (1971).PubMedCrossRefGoogle Scholar
  9. 9.
    Krahn, J., Stevens, F.C.: Biochemistry 11, 1804 (1972).PubMedCrossRefGoogle Scholar
  10. 10.
    Krahn, J., Stevens, F.C: Biochemistry 12, 1330 (1973).PubMedCrossRefGoogle Scholar
  11. 11.
    Stevens, F.C., Doskoch, E.: Canad.J. Biochem. 51, 1021 (1973).CrossRefGoogle Scholar
  12. 12.
    Krahn, J., Stevens, F.C.: FEBS Letters 28, 313 (1972).PubMedCrossRefGoogle Scholar
  13. 13.
    Odani, S., Ikenaka, T.: J. Biochem. 71, 839 (1972).PubMedGoogle Scholar
  14. 14.
    Odani, S., Koide, T., Ikenaka, T.: Proc. Japan Acad. 47, 621 (1971).Google Scholar
  15. 15.
    Seidl, D.S., Liener, J. E.: Biochim. biophys. Acta (Amst.) 251, 83 (1971).Google Scholar
  16. 16.
    Seidl, D.S., LIENERJ. E.: Biochim. biophys. Acta (Amst.) 258, 303 (1972).Google Scholar
  17. 17.
    Kowalski, D., Laskowski, M. Jr.: Biochemistry 11, 3451 (1972).PubMedCrossRefGoogle Scholar
  18. 18.
    Krahn, J.: Ph. D. thesis, University of Manitoba, 1973.Google Scholar
  19. 19.
    Wilson, K. A., LASKOWSKI, M., Sr.: J. biol. Chem. 248, 756 (1973).PubMedGoogle Scholar
  20. 20.
    Hixson, H.F., Jr., Laskowski, M. Jr.: Biochemistry 9, 166 (1970).PubMedCrossRefGoogle Scholar
  21. 21.
    Bidlingmeyer, U.D., Leary, T.R., Laskowski, M. Jr.: Biochemistry 11, 3303 (1973).CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1974

Authors and Affiliations

  • F. C. Stevens
    • 1
  • S. Wuerz
    • 1
  • J. Krahn
    • 1
  1. 1.Department of BiochemistryUniversity of ManitobaWinnipegCanada

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