Importance of tryptophan 49 of antithrombin in heparin binding and conformational activation.
Importance of tryptophan 49 of antithrombin in heparin binding and conformational activation.
复制标题
抗凝血酶色氨酸 49 在肝素结合和构象激活中的重要性。
DOI：
10.1021/bi050741i
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Desai,UmeshR
中科院分区：
文献类型：
--
作者：
Monien,BernhardH;Krishnasamy,Chandravel;Olson,StevenT;Desai,UmeshR
抗凝血酶的色氨酸49是凝血蛋白酶的主要抑制剂，以前通过化学修饰和诱变研究发现，它与变构激活剂肝素的结合有关。然而，抗凝血酶-五糖复合物的x射线共晶结构显示Trp49不与结合的糖接触。在这里，我们提供了肝素与抗凝血酶的Trp49 to Lys变体结合的详细热力学和动力学表征，并提出了Trp49如何参与肝素结合和激活的模型。Trp49突变为Lys导致肝素结合能在pH 7.4、I0.15和25°C下大量损失16 - 24%。这些损失是由于一个离子相互作用的损失（~ 30%）和非离子相互作用的损失（~ 70%）。快速动力学分析表明，突变对肝素与抗凝血酶的初始弱结合或随后蛇形蛋白构象激活的速率常数的影响最小。相反，突变的主要作用是使构象激活步骤逆转的速率常数增加70 - 100倍，从而使激活的构象不稳定。这种不稳定可能是由于涉及螺旋a的Trp49， Glu50和Lys53以及螺旋P的Ser112的相互作用网络的破坏，从而稳定了激活的构象。
Tryptophan 49 of antithrombin, the primary inhibitor of blood clotting proteinases, has previously been implicated in binding the allosteric activator, heparin, by chemical modification and mutagenesis studies. However, the X-ray cocrystal structure of the antithrombin−pentasaccharide complex shows that Trp49 does not contact the bound saccharide. Here, we provide a detailed thermodynamic and kinetic characterization of heparin binding to a Trp49 to Lys variant of antithrombin and suggest a model for how Trp49 participates in heparin binding and activation. Mutation of Trp49 to Lys resulted in substantial losses of 16−24% in heparin-binding energy at pH 7.4,I0.15, and 25 °C. These losses were due to both the loss of one ionic interaction (∼30%) and the loss of nonionic interactions (∼70%). Rapid kinetics analyses showed that the mutation minimally affected the initial weak binding of heparin to antithrombin or the rate constant for the subsequent conformational activation of the serpin. Rather, the principal effect of the mutation was to increase the rate constant for reversal of the conformational activation step by 70−100-fold, thereby destabilizing the activated conformation. This destabilization could be accounted for by the disruption of a network of interactions involving Trp49, Glu50, and Lys53 of helix A and Ser112 of helix P, which stabilizes the activated conformation.