Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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.
Koide, T., and Ikenaka, T. (1973) Eur. J. Biochem., 32, 417–431.
Yamamoto, M., Hara, S., and Ikenaka, T. (1983) J. Biochem., 94, 849–863.
Kouzuma, Y., Yamasaki, N., and Kimura, M. (1997) J. Biochem., 121, 456–463.
Yeh, K. W., Chen, J. C., Lin, M. I., Chen, Y. M., and Lin, C. Y. (1997) Plant Mol. Biol., 33, 565–570.
Wu, H. C., and Lin, J. Y. (1993) J. Biochem., 113, 258–263.
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.
McCoy, A. J., and Kortt, A. A. (1997) J. Mol. Biol., 269, 881–891.
Pouvreau, L., Chobert, J. M., Briand, L., Quillien, L., Tran, V., Gueguen, J., and Haertle, T. (1998) FEBS Lett., 423, 167–172.
Hung, C. H., Lee, M. C., and Lin, J. Y. (1994) FEBS Lett., 353, 312–314.
Song, S. I., Kim, C. H., Baek, S. J., and Choi, Y. D. (1993) Plant Physiol., 101, 1401–1402.
Yao, P. L., Hwang, M. J., Chen, Y. M., and Yeh, K. W. (2001) FEBS Lett., 496, 134–138.
Broze, G. J., Girard, T. J., and Novotny, W. F. (1990) Biochemistry, 29, 7539–7546.
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.
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.
Macedo, M. L., Garcia, V. A., Freire, M. G., and Richardson, M. (2007) Phytochemistry, 68, 1104–1111.
DiBella, F. P., and Liener, I. E. (1969) J. Biol. Chem., 244, 2824–2829.
Lin, J. Y., Chu, S. C., Wu, H. C., and Hsieh, Y. S. (1991) J. Biochem., 110, 879–883.
Krauchenco, S., Pando, S. C., Marangoni, S., and Polikarpov, I. (2003) Biochem. Biophys. Res. Commun., 26, 1303–1308.
Onesti, S., Brick, P., and Blow, D. M. (1991) J. Mol. Biol., 217, 153–176.
Sweet, R. M., Wright, H. T., Janim, J., Chotina, C. H., and Blow, D. M. (1974) Biochemistry, 13, 4212–4228.
Blow, D. M., Janin, J., and Sweet, R. M. (1974) Nature, 249, 54–57.
Barton, G. J. (1993) Protein Eng., 6, 37–40.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published in Russian in Biokhimiya, 2010, Vol. 75, No. 11, pp. 1577–1582.
Rights and permissions
About this article
Cite this article
Hung, CH., Chen, PJ. & Wang, HL. Evidence that Highly Conserved Residues of Delonix regia Trypsin Inhibitor Are Important for Activity. Biochemistry Moscow 75, 1388–1392 (2010). https://doi.org/10.1134/S0006297910110118
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0006297910110118