Vieira Saulo Martins, dos Reis Flávia Garcia, Geraldo Reinaldo, Dutra Denis Luis da Silva, Juliano Luiz, Julianod Maria Aparecida, Mignaco Julio Alberto, Zingali Russolina Benedeta
Instituto de Bioquímica Médica, UFRJ, Brasil.
Protein Pept Lett. 2013 Oct;20(10):1129-35. doi: 10.2174/09298665113209990001.
Thrombin, a highly specific protease of blood coagulation, has two exosites that modulate its specificity. We designed two sets of synthetic substrate FRET peptides with 25- or 11- amino acids (aa) each, based on the PAR 1 sequence, to characterize the effect of exosite 1 engagement on substrate catalysis and preference. The 25-aa set encompassed a sequence binding to exosite 1, and structural modeling showed that binding to thrombin did not differ significantly from that of PAR 1 peptide. Modification at the P3´position of the 25 or 11-aa peptides resulted in small effect on kinetic parameters. Ionic strength higher than physiologic depressed thrombin action on the 25-aa peptides. Addition of ligands of the exosite 1 negatively modulated the catalysis of 25-aa substrates. In conclusion, we succeeded to mimic and study in real time, using these synthetic peptides, the influence of ligand binding to exosite 1 on thrombin activity.
凝血酶是一种高度特异性的血液凝固蛋白酶，有两个外结合位点可调节其特异性。我们基于PAR 1序列设计了两组合成底物FRET肽，每组分别含有25个或11个氨基酸，以表征外结合位点1结合对底物催化作用和偏好性的影响。25氨基酸组包含与外结合位点1结合的序列，结构建模表明其与凝血酶的结合与PAR 1肽相比无显著差异。对25或11氨基酸肽的P3´位置进行修饰对动力学参数影响较小。高于生理水平的离子强度会抑制凝血酶对25氨基酸肽的作用。添加外结合位点1的配体会对25氨基酸底物的催化产生负调节作用。总之，我们成功地利用这些合成肽实时模拟并研究了配体与外结合位点1的结合对凝血酶活性的影响。
