Abstract
We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures εNH2-Cap-Leu-X-MCA and Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp, respectively. Abz (ortho-aminobenzoic acid) and EDDnp (2,4-dinitrophenyl-ethylenediamine) are the fluorescent donor-acceptor pair, and X was Cys(SBzl), Ser(OBzl), and Thr(OBzl) containing benzyl group (Bzl) at the functional side chain of Cys, Ser, and Thr. The peptidyl-coumarin-containing Cys(SBzl), Ser(OBzl), and Thr(OBzl) have higher affinity cathepsin B, supporting the interpretation of the crystal structure of rat cathepsin B complexed with the inhibitor Z-Arg-Ser(OBzl)-CH2Cl that the benzyl group attached to Ser hydroxyl side chain occupies the enzyme S′1 subsite [Jia et al. (1995), J. Biol. Chem. 270, 5527]. A similar effect of benzyl group was also detected in the internally quenched peptides. Finally, the benzyl group in substrates containing Cys(SBzl) amino acid at P1 seems to compensate the absence of adequate S2-P2 interaction in the hydrolysis of the peptides having Pro or Ala at P2 position.
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Alves, L. C., Almeida, P. C., Franzoni, L., Juliano, L., and Juliano, M. A. (1996). Peptide Res. 9, 92–96.
Authier, F., Posner, B. I., and Bergeron, J. J. (1996). FEBS Lett. 389, 55–60.
Barrett, A. J., Kemphave, A. A., Brown, M. A., Kirschke, H., Knight, C. G., Masaharu, T., and Hanada, K. (1982). Biochem. J. 201, 189–198.
Carmona, E., Dufour, É., Plouffe, C., Takebe, S., Manson, P., Mort, S. M., and Ménard, R. (1996). Biochemistry 35, 8149–8157.
Chagas, J. R., Juliano, L., and Prado, E. S. (1990). Anal. Biochem. 192, 419–425.
Del Nery, E., Juliano, M. A., Morten, M., Svendsen, I., Sharftstein, J., Walmsley, A. R., and Juliano, L. (1997). Biochem. J. 323, 427–433.
Duffy, M. J. (1992). Biotherapy 4, 45–52.
Esser, R. E., Angelo, R. A., Murphey, M. D., Watt, L. M., Thornburg, L. P., Palmer, J. T., Talhouk, J. W., and Smith. R. E. (1994). Arthritis Rheum. 37, 236–247.
Gauthier, F., Moreau, T., Lalmanach, G., Brillard-Bourdet, M., Di Martino, M. F., and Juliano, L. (1993). Arch. Biochem. Biophys. 306, 304–308.
Hasnain, S., Hirama, T., Huber, C. P., Mason, P., and Mort, J. S. (1993). J. Biol. Chem. 268, 235–240.
Hirata, I. Y., Cezari, M. H. S., Nakaie, C. R., Boschcov, P., Ito, A. S., Juliano, M. A., and Juliano, L. (1994). Lett. Peptide Sci. 1, 299–308.
Jia, Z., Hasnain, S., Hirama, T., Lee, X., Mort, J. S., To, R., and Huber, C. P. (1995). J. Biol. Chem. 270, 5527–5533.
Kakegawa, H., Nikawa, T., Tagami, K., Kamioka, H., Sumitani, K., Kawata, T., Drobnic-Kosorok, M., Lenarcic, B., Turk, V., and Katunuma, N. (1993). FEBS Lett. 321, 247–250.
Katumuna, N., Kominomi, E., and Li, K. (1987). Am. J. Pathol. 127, 681–688.
Kirchske, H., Wikstrom,. P., and Shaw, E. (1988). FEBS Lett. 228, 128–130.
Kirschke, H., Barrett, A. J., and Rawlings, N. D. (1995). Protein Profile 2, 1587–1644.
McGrath, M. E. (1999). Annu. Rev. Biophys. Biomol. Struct. 28, 181–204.
Ménard, R., and Storer, A. C. (1998). In Handbook of Proteolytic Enzymes (Barrett, A. J., Rawling, N. D., and Woesner, J. F.), Academic Press, New York, pp. 555–557.
Ménard, R., Khouri, H. E., Plouffe, C., Dupras, R., Rippoli, D., Vernet, T., Tessier, D. C., Laliberté, F., Thomas, D. Y., and Storer, A. C. (1990). Biochemistry 29, 6706–6713.
Musil, D., Zucic, D., Turk, D., Engh, R. A., Mayr, I., Huber, R., Popovic, T., Turk, V., Towatari, T., Katunuma, N., and Bode, W. (1991). EMBO J. 10, 2321–2330.
Portaro, F. C. V., Santos, B. F., Cezari, M. H. S., Juliano, M. A., Juliano, L., and Carmona, E. (2000). Biochem. J. (in press).
Schecter, I., and Berger, A. (1967). Biochem. Biophys. Res. Commun. 27, 157–162.
Shaw, E., Wikstrom, P., and Rusica, J. (1983). Arch. Biochem. Biophys. 222, 424–429.
Storer, A. C., and Ménard, R. (1994). Meth. Enzymol. 244, 486–500.
Wikstrom, P., and Ruscica, J. (1983). Arch. Biochem. Biophys. 222, 424–429.
Wilkinson, G. N. (1961). Biochem. J. 80, 324–332.
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Del Nery, E., Alves, L.C., Melo, R.L. et al. Specificity of Cathepsin B to Fluorescent Substrates Containing Benzyl Side-Chain-Substituted Amino Acids at P1 Subsite. J Protein Chem 19, 33–38 (2000). https://doi.org/10.1023/A:1007090708945
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DOI: https://doi.org/10.1023/A:1007090708945