Substrate-assisted catalysis of the PAR1 thrombin receptor - Enhancement of macromolecular association and cleavage
Substrate-assisted catalysis of the PAR1 thrombin receptor - Enhancement of macromolecular association and cleavage
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DOI：
10.1074/jbc.m004544200
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发表时间：
2000-12-29
影响因子：
4.8
通讯作者：
Kuliopulos, A
中科院分区：
文献类型：
--
作者：
Jacques, SL;LeMasurier, M;Kuliopulos, A
血小板活化和聚集是由凝血酶切割PAR1受体外域介导的。凝血酶对PAR1的特异性通过结合位于受体外域的水龙蛋白样区域（Hir）而增强。在这里，我们利用可溶性PAR1 n端外域通过稳态动力学测量来研究凝血酶对PAR1的识别和裂解机制。我们确定PAR1 Hir序列的主要作用是减少对接凝血酶-PAR1复合物形成的动力学障碍，而不是形成高亲和力的基态相互作用。此外，外源i结合的Hir基序促进了PARI (LDPR)-L-38/SFL44序列与凝血酶活性位点的有效相互作用，这一锁定过程是整个反应中能量最不利的步骤。随后的不可逆肽键裂解步骤是快速的，并且由于对接的Hir序列的存在而变弹性增强。此外，凝血酶裂解的c端外域产物，与活化受体相对应，与凝血酶紧密结合，这表明Hir序列在凝血酶活化受体中的另一个作用是将凝血酶隔离在血小板表面，并调节其他血小板受体的裂解，如PAR4凝血酶受体，后者缺乏功能性的Hir序列。
Platelet activation and aggregation are mediated by thrombin cleavage of the exodomain of the PAR1 receptor. The specificity of thrombin for PAR1 is enhanced by binding to a hirudin-like region (Hir) located in the receptor exodomain, Here, we examine the mechanism of thrombin-PAR1 recognition and cleavage by steady-state kinetic measurements using soluble PAR1 N-terminal exodomains. We determined that the primary role of the PAR1 Hir sequence is to reduce the kinetic barriers to formation of the docked thrombin-PAR1 complex rather than to form high affinity ground-state interactions. In addition, the exosite I-bound Hir motif facilitates the productive interaction of the PARI (LDPR)-L-38/SFL44 sequence with the active site of thrombin, This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin, This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence.