Le Bonniec B F, Guinto E R, Stone S R
Department of Haematology, University of Cambridge, England.
Biochemistry. 1995 Sep 26;34(38):12241-8. doi: 10.1021/bi00038a019.
The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1-antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin.
通过比较几种突变凝血酶与各种丝氨酸蛋白酶抑制剂（serpin）的结合速率常数（kon），研究了凝血酶催化凹槽在与丝氨酸蛋白酶抑制剂相互作用中的作用。结果表明，位于催化丝氨酸前三个残基处的Glu192以及与胰蛋白酶相比凝血酶序列中的主要插入片段（残基Tyr60A - Trp60D）在调节凝血酶与丝氨酸蛋白酶抑制剂的相互作用中起重要作用。用谷氨酰胺取代Glu192使与α1 - 抗胰蛋白酶（其P1位为甲硫氨酸）的kon值增加了3个数量级，但对含有P1位精氨酸的丝氨酸蛋白酶抑制剂的kon值没有明显改变。缺失PPW的凝血酶突变体（缺少残基Pro60B、Pro60C和Trp60D）与蛋白酶连接蛋白-1的kon值与凝血酶相似，但与抗凝血酶III的kon值低2个数量级。因此，凝血酶的60环插入对于其与抗凝血酶III的相互作用似乎至关重要，但对于与蛋白酶连接蛋白-1形成复合物则是可有可无的。肝素显著增加了所有测试凝血酶变体与抗凝血酶III和蛋白酶连接蛋白-1的kon值，但在缺少残基Glu146、Thr147和Trp148（相对于60环插入位于催化位点另一侧）的凝血酶突变体（des - ETW）中观察到更显著的效果。在最佳浓度下，肝素使des - ETW - 抗凝血酶III相互作用的kon值增加了近5个数量级，比凝血酶增加的幅度大得多，这表明肝素能够部分补偿des - ETW突变对凝血酶结构的不利影响。
