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|Title:||Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin|
|Authors:||Pimenta, D. C.|
Juliano, M. A.
Universidade Federal de São Paulo (UNIFESP)
|Citation:||Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology. Amsterdam: Elsevier B.V., v. 1544, n. 1-2, p. 113-122, 2001.|
|Abstract:||Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P-2 and P-2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp = N-[2,4-dinitrophenyl]-ethylenediamine and Abz = orthu-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low K-m values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (K-m = 0.27 muM, k(cat) = 16.15 s(-1), k(cat)/K-m = 60185 muM(-1) s(-1)). (C) 2001 Elsevier Science B.V. All rights reserved.|
|Appears in Collections:||Artigo|
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