Biochemistry (Moscow)

, Volume 75, Issue 11, pp 1388–1392 | Cite as

Evidence that Highly Conserved Residues of Delonix regia Trypsin Inhibitor Are Important for Activity

  • Chih-Hung HungEmail author
  • Pei-Jung Chen
  • Hai-Lung Wang


Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89) is not required.

Key words

Delonix regia trypsin inhibitor Kunitz-type trypsin inhibitor Delonix regia reactive site site-directed mutagenesis 


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  1. 1.
    Pando, S. C., Oliva, M. L. V., Sampaio, C. A. M., Ciero, L. Di Novello, J. C., and Marangoni, S. (2001) Phytochemistry, 57, 625–631.CrossRefPubMedGoogle Scholar
  2. 2.
    Koide, T., and Ikenaka, T. (1973) Eur. J. Biochem., 32, 417–431.CrossRefPubMedGoogle Scholar
  3. 3.
    Yamamoto, M., Hara, S., and Ikenaka, T. (1983) J. Biochem., 94, 849–863.PubMedGoogle Scholar
  4. 4.
    Kouzuma, Y., Yamasaki, N., and Kimura, M. (1997) J. Biochem., 121, 456–463.PubMedGoogle Scholar
  5. 5.
    Yeh, K. W., Chen, J. C., Lin, M. I., Chen, Y. M., and Lin, C. Y. (1997) Plant Mol. Biol., 33, 565–570.CrossRefPubMedGoogle Scholar
  6. 6.
    Wu, H. C., and Lin, J. Y. (1993) J. Biochem., 113, 258–263.PubMedGoogle Scholar
  7. 7.
    Hung, C. H., Peng, P. H., Huang, C. C., Wang, H. L., Chen, Y. J., Chen, Y. L., and Chi, L. M. (2007) Biosci. Biotechnol. Biochem., 71, 98–103.CrossRefPubMedGoogle Scholar
  8. 8.
    McCoy, A. J., and Kortt, A. A. (1997) J. Mol. Biol., 269, 881–891.CrossRefPubMedGoogle Scholar
  9. 9.
    Pouvreau, L., Chobert, J. M., Briand, L., Quillien, L., Tran, V., Gueguen, J., and Haertle, T. (1998) FEBS Lett., 423, 167–172.CrossRefPubMedGoogle Scholar
  10. 10.
    Hung, C. H., Lee, M. C., and Lin, J. Y. (1994) FEBS Lett., 353, 312–314.CrossRefPubMedGoogle Scholar
  11. 11.
    Song, S. I., Kim, C. H., Baek, S. J., and Choi, Y. D. (1993) Plant Physiol., 101, 1401–1402.CrossRefPubMedGoogle Scholar
  12. 12.
    Yao, P. L., Hwang, M. J., Chen, Y. M., and Yeh, K. W. (2001) FEBS Lett., 496, 134–138.CrossRefPubMedGoogle Scholar
  13. 13.
    Broze, G. J., Girard, T. J., and Novotny, W. F. (1990) Biochemistry, 29, 7539–7546.CrossRefPubMedGoogle Scholar
  14. 14.
    Do Socorro, M. C. M., Oliva, M. L., Fritz, H., Jochum, M., Mentele, R., Sampaio, M., Coelho, L. C., Batista, I. F., and Sampaio, C. A. (2002) Biochem. Biophys. Res. Commun., 291, 635–639.CrossRefGoogle Scholar
  15. 15.
    Araujo, A. P., Hansen, D., Vieira, D. F., Oliveira, C., Santana, L. A., Beltramini, L. M., Sampaio, C. A., Sampaio, M. U., and Oliva, M. L. (2005) Biol. Chem., 386, 561–568.CrossRefPubMedGoogle Scholar
  16. 16.
    Macedo, M. L., Garcia, V. A., Freire, M. G., and Richardson, M. (2007) Phytochemistry, 68, 1104–1111.CrossRefPubMedGoogle Scholar
  17. 17.
    DiBella, F. P., and Liener, I. E. (1969) J. Biol. Chem., 244, 2824–2829.PubMedGoogle Scholar
  18. 18.
    Lin, J. Y., Chu, S. C., Wu, H. C., and Hsieh, Y. S. (1991) J. Biochem., 110, 879–883.PubMedGoogle Scholar
  19. 19.
    Krauchenco, S., Pando, S. C., Marangoni, S., and Polikarpov, I. (2003) Biochem. Biophys. Res. Commun., 26, 1303–1308.CrossRefGoogle Scholar
  20. 20.
    Onesti, S., Brick, P., and Blow, D. M. (1991) J. Mol. Biol., 217, 153–176.CrossRefPubMedGoogle Scholar
  21. 21.
    Sweet, R. M., Wright, H. T., Janim, J., Chotina, C. H., and Blow, D. M. (1974) Biochemistry, 13, 4212–4228.CrossRefPubMedGoogle Scholar
  22. 22.
    Blow, D. M., Janin, J., and Sweet, R. M. (1974) Nature, 249, 54–57.CrossRefPubMedGoogle Scholar
  23. 23.
    Barton, G. J. (1993) Protein Eng., 6, 37–40.CrossRefPubMedGoogle Scholar

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© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  1. 1.Department of Medical Laboratory Science and BiotechnologyYuanpei UniversityHsinchu 300Taiwan, ROC

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