Specificity of human tissue kallikrein towards substrates containing Phe-Phe pair of amino acids

Specificity of human tissue kallikrein towards substrates containing Phe-Phe pair of amino acids

Author Pimenta, Daniel C. Google Scholar
Chao, Julie Google Scholar
Chao, Lee Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Med Univ S Carolina
Abstract We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK(1)) of substrates containing the Phe-Phe amino acid pair, after which hK(1) cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1-14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P-9 to P'(13), and the kinetic parameters of their hydrolysis by hK(1) were determined. All these peptides were cleaved after the Phe-Phe pair. for comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe-Arg and Leu-Lys bonds, respectively. Hybrid peptides containing kallistatin-PCI sequences showed that the efficiency of hK(1) activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P-1 amino acid as well as on residues at the P- and PI-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe-Phe, and on Lys and lie at the P-3 and P-4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp, All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe-Arg pair; however, hydrolysis after Phe-Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme-substrate interaction and/or particular substrate conformations.
Keywords fluorescent substrate
Language English
Date 1999-04-15
Published in Biochemical Journal. London: Portland Press, v. 339, p. 473-479, 1999.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press
Extent 473-479
Origin http://dx.doi.org/10.1042/0264-6021:3390473
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000079918400033
URI http://repositorio.unifesp.br/handle/11600/26065

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