Parry M A, Myles T, Tschopp J, Stone S R
Department of Haematology, University of Cambridge, U.K.
Biochem J. 1996 Nov 15;320 ( Pt 1)(Pt 1):335-41. doi: 10.1042/bj3200335.
The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38-60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR39-40) by thrombin were 107 s-1 and 1.3 microM; the kcat/Km of TR39-40 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR39-40 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR39-40 efficiently (kcat/Km > 10(6) M-1.s-1). Whereas trypsin cleaved TR39-40 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR39-40 by chymotrypsin (kcat/Km approximately 10(6) M-1.s-1) was predominantly due to a low Km value (2.8 microM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR39-40 at the Arg41-Ser43 bond, but exhibited kcat/Km values that were at least 10(3)-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR39-60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/K(m) value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
测定了凝血酶和其他12种蛋白酶对基于凝血酶受体激活位点（38 - 60位氨基酸残基）的肽段进行水解的动力学参数。凝血酶切割该肽段（TR39 - 40）的kcat和Km值分别为107 s-1和1.3 μM；TR39 - 40的kcat/Km是凝血酶所观察到的最高值之一。提出了一个模型，该模型将肽段切割参数与细胞对凝血酶反应的浓度依赖性进行了协调。TR39 - 40的切割并非凝血酶所特有。胰腺蛋白酶胰蛋白酶和胰凝乳蛋白酶能有效水解TR39 - 40（kcat/Km > 10(6) M-1.s-1）。胰蛋白酶在凝血酶激活位点（Arg41 - Ser42）切割TR39 - 40，而胰凝乳蛋白酶在Phe43之后水解该肽段。这种胰凝乳蛋白酶切割会导致受体失活。胰凝乳蛋白酶对TR39 - 40的有效切割（kcat/Km约为10(6) M-1.s-1）主要归因于低Km值（2.8 μM）。蛋白酶因子Xa、纤溶酶、血浆激肽释放酶、活化蛋白C和颗粒酶A也在Arg41 - Ser43键处水解TR39 - 40，但它们的kcat/Km值比凝血酶至少低10(3)倍。组织型和尿激酶型纤溶酶原激活剂以及颗粒酶B和中性粒细胞弹性蛋白酶都不能以可观的速率切割TR39 - 60。然而，中性粒细胞组织蛋白酶G在Phe55之后水解受体肽段。与胰凝乳蛋白酶切割一样，这种切割会导致受体失活，但组织蛋白酶G反应的效率明显较低；kcat/K(m)值比凝血酶低近四个数量级。除上述切割位点外，在Arg46处还鉴定出凝血酶和其他精氨酸特异性蛋白酶的一个次要切割位点，但该位点的切割仅以非常低的速率发生，在体内不太可能具有重要意义。
