title: Human tissue kallikrein S1 subsite recognition of non-natural basic amino acids.
abstract: We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [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 stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100-São Paulo 04044-020, Brazil.
authors.forename: R L
authors.initials: RL
authors.lastname: Melo
authors.forename: R C
authors.initials: RC
authors.lastname: Barbosa Pozzo
authors.forename: D C
authors.initials: DC
authors.lastname: Pimenta
authors.forename: E
authors.initials: E
authors.lastname: Perissutti
authors.forename: G
authors.initials: G
authors.lastname: Caliendo
authors.forename: V
authors.initials: V
authors.lastname: Santagada
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000138:chemical synthesis* / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000097:blood; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D013379:Substrate Specificity; D020840:Tissue Kallikreins / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010649:Phenylalanine; D006639:Histidine; D001120:Arginine; D007610:Kallikreins; D020840:Tissue Kallikreins; D014357:Trypsin