Melo R L, Barbosa Pozzo R C, Pimenta D C, Perissutti E, Caliendo G, Santagada V, Juliano L, Juliano M A
Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100-São Paulo 04044-020, Brazil.
Biochemistry. 2001 May 1;40(17):5226-32. doi: 10.1021/bi002003u.
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.
我们使用基于缓激肽C末端区域人激肽原序列的内淬灭荧光肽Abz-F-X-S-R-Q-EDDnp和Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz为邻氨基苯甲酸；EDDnp为N-(2,4-二硝基苯基)乙二胺]，探究了人尿激肽释放酶（hK1）主要S(1)亚位点独特的底物特异性，该亚位点可同时接受苯丙氨酸（Phe）和精氨酸（Arg）。在天然序列中代表Arg的X位点，被替换为以下合成的碱性非天然氨基酸：4-(氨基甲基)苯丙氨酸（Amf）、4-胍基苯丙氨酸（Gnf）、4-(氨基甲基)-N-异丙基苯丙氨酸（Iaf）、N(im)-(二甲基)组氨酸 [H(2Me)]、3-吡啶基丙氨酸（Pya）、4-哌啶基丙氨酸（Ppa）、4-(氨基甲基)环己基丙氨酸（Ama）和4-(氨基环己基)丙氨酸（Aca）。只有Abz-F-Amf-S-R-Q-EDDnp和Abz-F-H(2Me)]-S-R-Q-EDDnp能被有效水解，其他所有肽均对水解具有抗性。然而，Abz-F-Ama-S-R-Q-EDDnp以50 nM的抑制常数（K(i)）抑制hK1，与人类血浆激肽释放酶、凝血酶、纤溶酶和胰蛋白酶相比，具有高度特异性。Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp系列对hK1更敏感，尽管含有Gnf、Pya和Ama的肽对其具有抗性。出乎意料的是，X为组氨酸（His）、赖氨酸（Lys）、H(2Me)、Amf、Iaf、Ppa和Aca的肽在这些氨基酸的氨基或羧基位点被切割，表明S(1)'亚位点对碱性残基有显著偏好。除了X为Arg的天然序列外，人类血浆激肽释放酶不水解该系列的任何肽。总之，hK1的S(1)亚位点接受具有碱性和芳香族侧链组合的氨基酸，尽管对于S(1)-P(1)相互作用，其偏好脂肪族和碱性侧链。
