Engineering Thrombin for Selective Specificity toward Protein C and PAR1
Engineering Thrombin for Selective Specificity toward Protein C and PAR1
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DOI：
10.1074/jbc.m110.119875
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发表时间：
2010-06-18
影响因子：
4.8
通讯作者：
Di Cera, Enrico
中科院分区：
文献类型：
--
作者：
Marino, Francesca;Pelc, Leslie A.;Di Cera, Enrico
凝血酶通过与多种生理底物相互作用，引起对血液动态平衡至关重要的功能反应。对97个残基进行ALA扫描突变，发现Trp(215)是凝血酶特异性的一个最重要的决定因素。Trp(215)的饱和突变产生了具有k(Cat)/K-m值的结构，用于纤维蛋白原、蛋白酶激活的受体PAR1和蛋白C的水解性，跨越五个数量级。重要的是，Trp(215)替换的效果依赖于上下文。突变体W215E对蛋白C的特异性是纤维蛋白原和PAR1的10倍，这代表了相对于野生型的显著变化，即对纤维蛋白原和PAR1的特异性比蛋白C高100倍。然而，当W215E突变与自溶环中9个残基的缺失相结合时，这本身就将酶的特异性从纤维蛋白原和PAR1转移到蛋白C，所得到的结构只对PAR1具有显著的活性。这些发现表明，凝血酶可以被重新设计为对蛋白C和PAR1的选择性特异性。Trp(215)突变为剖析凝血酶在血液中的多功能作用和临床应用提供了重要的试剂。
Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp(215) as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp(215) produces constructs featuring k(cat)/K-m values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and protein C that span five orders of magnitude. Importantly, the effect of Trp(215) replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp(215) provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications.