Underwood M C, Zhong D, Mathur A, Heyduk T, Bajaj S P
Department of Biochemistry and Molecular Biology and the Departments of Medicine and Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
J Biol Chem. 2000 Nov 24;275(47):36876-84. doi: 10.1074/jbc.M001386200.
The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.
凝血因子Xa（FXa）的丝氨酸蛋白酶结构域含有一个钠结合位点和一个钙结合位点。在此，我们研究了这两个阳离子结合位点的功能意义及其与S1位点的热力学联系。动力学数据表明，在不存在Ca(2+)时，Na(+)以K(d)约39 mM的亲和力结合到与底物结合的FXa上，而在存在Ca(2+)时，该亲和力约为9.5 mM。结合钠的FXa（钠-FXa）在水解S-2222（苯甲酰-Ile-Glu-Gly-Arg-对硝基苯胺）时的催化效率提高了约18倍（K(m)约降低4.5倍，k(cat)约增加4倍），并且Ca(2+)进一步使该k(cat)增加约1.4倍。在不存在Na(+)时，Ca(2+)以K(d)约705 μM的亲和力结合到底物结合的FXa的蛋白酶结构域，而在存在Na(+)时，该亲和力约为175 μM。Ca(2+)结合到FXa的蛋白酶结构域（FXa-钙）对K(m)没有影响，但在水解S-2222时使k(cat)增加约4倍，并且Na(+)进一步使该k(cat)增加约1.4倍。与K(m)数据一致，钠-FXa在与对氨基苯甲脒（S1位点探针）相互作用时的亲和力增加了约5倍，在与双结构域组织因子途径抑制剂结合时的速率增加了约4倍；Ca(2+)（无论有无Na(+)）对这些相互作用均无影响。抗凝血酶以约快4倍的速率结合到FXa-钙上，以约快24倍的速率结合到钠-FXa上，以约快28倍的速率结合到钠-FXa-钙上。因此，Ca(2+)和Na(+)共同使FXa的催化效率提高了约28倍。Na(+)增强Ca(2+)的结合，而Ca(2+)增强Na(+)的结合。此外，Na(+)增强S1位点的占据，而S1位点的占据增强Na(+)的结合。因此，Na(+)位点在热力学上与S1位点以及蛋白酶结构域的Ca(2+)位点相联系，而Ca(2+)位点仅与Na(+)位点相联系。这些发现的意义在于，在生理性凝血过程中，形成的大多数FXa将以具有最大生物活性的钠-FXa-钙的形式存在。
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