Human tissue kallikrein S-1 subsite recognition of non-natural basic aminoacids

We explored the unique substrate specificity of the primary S-1 subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using inter nally quenched fluorescent peptides Abz-G-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X -S-S-R-P-B-EBBnp [Abz is o-aminobenzoic acid, EDDnp is N-(2,4-dinitrophenyl )ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin, Position X, which in natural sequence stan ds for Arg; received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(am inomethyl)-N-isopropylphenylala (Iaf), N-im-(dimethyl)histidine [H(2Me)], 3 -pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(atninomethyl)cyclohex ylalnnine (Ama), and 4-(aminocyclohexyl)alanine (Aca), Only Abz-F-Amf-S-R-Q -EDDnp and Abz-F-[H(2Me)]-S-R-B-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EBDnp inhibit ed hK1 with a K-i of 50 nM with high specificity compared to human plasma k allikrein, thrombin, plasmin, and trypsin, The Abz-G-F-S-P-F-X-S-S-R-P-B-ED Dnp series were more susceptible to hK1, although the peptides with Gnf, Pp a, and Ama were resistant to it. Unexpectedly, the peptides in which X is H is, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxy l sires of these amino acids, indicating that the S-1' subsite has signific ant preference for basic residues. Human plasma kallikrein did not hydrolyz e any peptide of this series except the natural sequence where X is Arg. In conclusion, the Si subsite of hK1 accepts amino acids with combined basic anal aromatic side chain, although for the S-1-P-1 interaction the preferen ce is for aliphatic and basic side chains.