Camire Rodney M
Joseph Stokes, Jr. Research Institute, Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2002 Oct 4;277(40):37863-70. doi: 10.1074/jbc.M203692200. Epub 2002 Jul 30.
Two loop segments (183-189 and 221-225) in the protease domain of factor Xa contribute to the formation of a Na(+)-binding site. Studies with factor Xa indicate that binding of a single Na(+) ion to this site influences its activity by altering the S1 specificity site, and substitution of Tyr(225) with Pro diminishes sensitivity to Na(+). Using full-length factor Xa(Y225P), the allosteric relationship between the Na(+) site and other structural determinants in factor Xa and prothrombinase was investigated. Direct binding and kinetic measurements with probes that target the S1 specificity pocket indicate that assembly of the mutant in prothrombinase corrected the impaired binding of these probes observed with free factor Xa(Y225P). This appears to result from the apparent allosteric linkage between the factor Va, S1, and Na(+)-binding sites, since binding of the cofactor to membrane-bound factor Xa(Y225P) enhances binding at the S1 site and vice versa. Additional studies revealed that the internal salt bridge (Ile(16)-Asp(194)) of factor Xa(Y225P) is partially destabilized, a process that is reversible upon occupation of the S1 site. The data establish that alterations at the factor Xa Na(+)-binding site shift the zymogen-protease equilibrium to a more zymogen-like state, and as a consequence binding of S1-directed probes and factor Va are adversely affected. Therefore, the zymogen-like characteristics of factor Xa(Y225P) have allowed for the apparent allosteric linkage between the S1, factor Va, and Na(+) sites to become evident and has provided insight into the structural transitions which accompany the conversion of factor X to factor Xa.
凝血因子Xa蛋白酶结构域中的两个环段（183 - 189和221 - 225）有助于形成一个钠离子结合位点。对凝血因子Xa的研究表明，单个钠离子与该位点的结合通过改变S1特异性位点来影响其活性，用脯氨酸取代酪氨酸（225）会降低对钠离子的敏感性。利用全长凝血因子Xa（Y225P），研究了凝血因子Xa和凝血酶原酶中钠离子位点与其他结构决定因素之间的变构关系。使用靶向S1特异性口袋的探针进行直接结合和动力学测量表明，凝血酶原酶中突变体的组装纠正了游离凝血因子Xa（Y225P）中观察到的这些探针结合受损的情况。这似乎是由于辅因子Va、S1和钠离子结合位点之间明显的变构联系，因为辅因子与膜结合的凝血因子Xa（Y225P）的结合增强了S1位点的结合，反之亦然。进一步的研究表明，凝血因子Xa（Y225P）的内部盐桥（异亮氨酸（16）-天冬氨酸（194））部分不稳定，该过程在S1位点被占据后是可逆的。数据表明，凝血因子Xa钠离子结合位点的改变将酶原 - 蛋白酶平衡转变为更类似酶原的状态，结果是S1导向探针和辅因子Va的结合受到不利影响。因此，凝血因子Xa（Y225P）的酶原样特征使得S1、辅因子Va和钠离子位点之间明显的变构联系变得明显，并为凝血因子X向凝血因子Xa转化过程中伴随的结构转变提供了见解。
