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|Title:||Human tissue kallikrein S-1 subsite recognition of non-natural basic amino acids|
|Authors:||Melo, R. L.|
Pimenta, D. C.
Juliano, M. A.
Universidade Federal de São Paulo (UNIFESP)
Univ Naples Federico II
|Publisher:||Amer Chemical Soc|
|Citation:||Biochemistry. Washington: Amer Chemical Soc, v. 40, n. 17, p. 5226-5232, 2001.|
|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-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 stands for Arg; received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylala (Iaf), N-im-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(atninomethyl)cyclohexylalnnine (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 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-B-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Ppa, 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 sires 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 Si subsite of hK1 accepts amino acids with combined basic anal aromatic side chain, although for the S-1-P-1 interaction the preference is for aliphatic and basic side chains.|
|Appears in Collections:||Artigo|
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