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

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.