Specificity of human tissue kallikrein towards substrates containing Phe-Phe pair of amino acids.
We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe-Phe amino acid pair, after which hK1 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 P9 to P'13, and the kinetic parameters of their hydrolysis by hK1 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 hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P'-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe-Phe, and on Lys and Ile at the P3 and P4 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.
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Departamento de Biof approximately ísica, Escola Paulista de Medicina-UNIFESP, Rua Três de Maio, 100-São Paulo, 04044-020, Brazil.
Chao
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Juliano
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Juliano
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D000595:Amino Acid Sequence; D000818:Animals; D004151:Dipeptides / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D020840:Tissue Kallikreins
D004151:Dipeptides; D010649:Phenylalanine; D007610:Kallikreins; D020840:Tissue Kallikreins