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Biochemistry (Moscow)

, Volume 73, Issue 10, pp 1140–1145 | Cite as

Leu254 residue and calcium ions as new structural determinants of carboxypeptidase T substrate specificity

  • A. M. Grishin
  • V. Kh. AkparovEmail author
  • G. G. Chestukhina
Article

Abstract

New determinants of Thermoactinomyces vulgaris carboxypeptidase T (CPT) substrate specificity—structural calcium ions and Leu254 residue—were found by means of steady-state kinetics and site-directed mutagenesis. The removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64/1.7/1 to 162/1.3/1. Substitution of the hydrophobic Leu254 in CPT for polar Asn did not change hydrolysis efficiency of substrates with C-terminal Leu and Arg, but resulted in more than 28-fold decrease in activity towards the substrate with C-terminal Glu. It is shown that the His68 residue is not a structural determinant of CPT specificity.

Key words

carboxypeptidase T substrate specificity rational redesign protein engineering site-directed mutagenesis 

Abbreviations

CPA

carboxypeptidase A

CPB

carboxypeptidase B

CPT

carboxypeptidase T

CHAPS

3-[(3-cholamidopropyl)dimethylammonium]-1-propane sulfate

DnpAAR

2,4-dinitrophenyl-alanyl-alanyl-arginine

MCP

metallocar-boxypeptidase

ZAAL

benzyloxycarbonyl-alanyl-alanyl-leucine

ZAAE

benzyloxycarbonyl-alanyl-alanyl-glutamic acid

ZAALpNA

benzyloxycarbonyl-alanyl-alanyl-leucine p-nitroanilide

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References

  1. 1.
    Osterman, A. L., Stepanov, V. M., Rudenskaya, G. N., Khodova, O. M., and Tsaplina, I. A. (1984) Biokhimiya, 49, 292–301.Google Scholar
  2. 2.
    Auld, D. S. (1997) in Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds.) Academic Press, Inc., N.Y., pp. 1321–1328.Google Scholar
  3. 3.
    Aviles, F. X., and Vendrell, J. (1997) in Handbook of Proteolytic Enzymes (Barrett, A. J., Rawlings, N. D., and Woessner, J. F., eds.) Academic Press, Inc., N.Y., pp. 1333–1335.Google Scholar
  4. 4.
    Teplyakov, A., Polyakov, K., Obmolova, G., Strokopytov, B., Kuranova, I., Osterman, A., Grishin, N., Smulevitch, S., Zagnitko, O., Galperina, O., Matz, M., and Stepanov, V. M. (1992) Eur. J. Biochem., 208, 281–288.PubMedCrossRefGoogle Scholar
  5. 5.
    Kim, H., and Lipscomb, W. N. (1991) Biochemistry, 30, 8171–8180.PubMedCrossRefGoogle Scholar
  6. 6.
    Adler, M., Bryant, J., Buckman, B., Islam, I., Larsen, B., Finster, S., Kent, L., May, K., Mohan, R., Yuan, S., and Whitlow, M. (2005) Biochemistry, 44, 9339–9347.PubMedCrossRefGoogle Scholar
  7. 7.
    Reeck, G. R., Walsh, K. A., Hermodson, M. A., and Neurath, H. (1971) Proc. Natl. Acad. Sci. USA, 68, 1226–1230.PubMedCrossRefGoogle Scholar
  8. 8.
    Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C. B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem., 263, 17828–17836.PubMedGoogle Scholar
  9. 9.
    Osterman, A. L., Grishin, N. V., Smulevitch, S. V., Matz, M. V., Zagnitko, O. P., Revina, L. P., and Stepanov, V. M. (1992) J. Protein Chem., 11, 561–570.PubMedCrossRefGoogle Scholar
  10. 10.
    Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun., 27, 157–162.PubMedCrossRefGoogle Scholar
  11. 11.
    Wolff, E. C., Schirmer, E. W., and Folk, J. E. (1962) J. Biol. Chem., 237, 3094–3099.PubMedGoogle Scholar
  12. 12.
    Edge, M., Forder, C., Hennam, J., Lee, I., Tonge, D., Hardern, I., Fitton, J., Eckersley, K., East, S., Shufflebotham, A., Blakey, D., and Slater, A. (1998) Protein Eng., 11, 1229–1234.PubMedCrossRefGoogle Scholar
  13. 13.
    Akparov, V. Kh., Grishin, A. M., Yusupova, M. P., Ivanova, N. M., and Chestukhina, G. G. (2007) Biochemistry (Moscow), 72, 416–423.PubMedGoogle Scholar
  14. 14.
    Trachuk, L. A., Bushueva, A. M., Shevelev, A. B., Novgorodova, S. A., Akparov, V. Kh., and Chestukhina, G. G. (2002) Vopr. Med. Khim., 48, 577–585.PubMedGoogle Scholar
  15. 15.
    Novagen pET System Manual TB055, 7th Edn. (1997) Novagen Madison, W.I.Google Scholar
  16. 16.
    Smulevitch, S. V., Osterman, A. L., Galperina, O. V., Matz, M. V., Zagnitko, O. P., Kadyrov, R. M., Tsaplina, I. A., Grishin, N. V., Chestukhina, G. G., and Stepanov, V. M. (1991) FEBS Lett., 291, 75–78.PubMedCrossRefGoogle Scholar
  17. 17.
    Lyublinskaya, L. A., Yakusheva, L. D., and Stepanov, V. M. (1977) Bioorg. Khim., 3, 273–279.Google Scholar
  18. 18.
    Yusupova, M. P., Kotlova, E. K., Timokhina, E. A., and Stepanov, V. M. (1995) Bioorg. Khim., 21, 33–38.Google Scholar
  19. 19.
    Voyushina, T. L., Lyublinskaya, L. A., Timokhina, E. A., and Stepanov, V. M. (1987) Bioorg. Khim., 13, 615–622.Google Scholar
  20. 20.
    Cueni, L. B., Bazzone, T. J., Riordan, J. F., and Vallee, B. L. (1980) Analyt. Biochem., 107, 341–349.PubMedCrossRefGoogle Scholar
  21. 21.
    Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene, 77, 51–59.PubMedCrossRefGoogle Scholar
  22. 22.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, NY.Google Scholar
  23. 23.
    Trachuk, L., Letarov, A., Kudelina, I. A., Yusupova, M. P., and Chestukhina, G. G. (2005) Protein Exp. Purif., 40, 51–59.CrossRefGoogle Scholar
  24. 24.
    Bradford, M. M. (1976) Analyt. Biochem., 72, 248–254.PubMedCrossRefGoogle Scholar
  25. 25.
    Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) Protein Sci., 4, 2411–2423.PubMedCrossRefGoogle Scholar
  26. 26.
    Laemmli, U. K. (1970) Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  27. 27.
    Klesov, A. A., and Vallee, B. L. (1977) Bioorg. Khim., 3, 806–816.Google Scholar
  28. 28.
    Zisapel, N., Kurn-Abramowitz, N., and Sokolovsky, M. (1973) Eur. J. Biochem., 35, 507–511.PubMedCrossRefGoogle Scholar
  29. 29.
    Christianson, D. W., and Lipscomb, W. N. (1987) J. Am. Chem. Soc., 109, 5536–5538.CrossRefGoogle Scholar
  30. 30.
    Auld, D. S., and Vallee, B. L. (1970) Biochemistry, 9, 602–609.PubMedCrossRefGoogle Scholar
  31. 31.
    Bayes, A., Fernandez, D., Sola, M., Marrero, A., Garcia-Pique, S., Aviles, F. X., Vendrell, J., and Gomis-Ruth, F. X. (2007) Biochemistry, 46, 6921–6930.PubMedCrossRefGoogle Scholar
  32. 32.
    Spungin, A., and Blumberg, S. (1989) Eur. J. Biochem., 183, 471–477.PubMedCrossRefGoogle Scholar
  33. 33.
    Nalamachu, S. R., Song, L., and Fricker, L. D. (1994) J. Biol. Chem., 269, 11192–11195.PubMedGoogle Scholar
  34. 34.
    Perona, J. J., and Craik, C. S. (1995) Protein Sci., 4, 337–360.PubMedCrossRefGoogle Scholar
  35. 35.
    Stepanov, V. M. (1995) in Methods in Enzymology (Barrett, A. J., ed.) Vol. 248, Academic Press, Inc., N.Y., pp. 675–683.Google Scholar

Copyright information

© MAIK Nauka 2008

Authors and Affiliations

  • A. M. Grishin
    • 1
  • V. Kh. Akparov
    • 1
    Email author
  • G. G. Chestukhina
    • 1
  1. 1.Institute of Genetics and Selection of Industrial MicroorganismsMoscowRussia

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