Rudolph A E, Porche-Sorbet R, Miletich J P
Departments of Pathology and Medicine, Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
J Biol Chem. 2001 Feb 16;276(7):5123-8. doi: 10.1074/jbc.M006961200. Epub 2000 Nov 21.
We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fX(R306A), fX(E310N), fX(R347N), fX(K351A), and fX(K414A), are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa(E310N), which has a reduced apparent affinity ( approximately 3-fold) for prothrombin compared with wild type fXa (fXa(WT)). In the absence of phospholipid, fVa enhances the catalytic activity of fXa(WT) significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXa(WT), fXa(R306A), and fXa(E310N) similarly, whereas fXa(R347N), fXa(K351A), and fXa(K414A) demonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXa(WT) on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.
我们之前报道过，活化的因子X（fXa）中的347位氨基酸残基有助于辅因子因子Va（fVa）的结合（鲁道夫，A.E.，波切-索贝，R.和米莱蒂奇，J.P.（2000年）《生物化学》39卷，2861 - 2867页）。现在通过诱变鉴定出了另外四个参与fVa结合的氨基酸残基。所有五个产生的fX变体，fX(R306A)、fX(E310N)、fX(R347N)、fX(K351A)和fX(K414A)，都能正常活化和被抑制。然而，在次最大浓度肝素存在下，抗凝血酶III对所有这些酶的抑制速率都降低了。在没有fVa的情况下，除了fXa(E310N)外，所有酶与凝血酶原的结合和活化方式相似，与野生型fXa（fXa(WT)）相比，fXa(E310N)对凝血酶原的表观亲和力降低（约3倍）。在没有磷脂的情况下，fVa能显著增强fXa(WT)的催化活性，但变体酶的反应大大减弱。加入100 nM的PC:PS（3:1）脂质体后，fVa对fXa(WT)、fXa(R306A)和fXa(E310N)的增强作用相似，而在这些条件下，fXa(R347N)、fXa(K351A)和fXa(K414A)表现出接近正常的催化活性，但对fVa的表观亲和力降低。所有酶在活化血小板上的功能与fXa(WT)相似，活化血小板在理想表面上能提供饱和的fVa。通过竞争结合试验证实了由于Arg - 347、Lys - 351和Lys - 414位点的取代导致对fVa结合亲和力的丧失。因此，Arg - 347、Lys - 351和Lys - 414可能是fVa核心结合位点的一部分，而Arg - 306和Glu - 310的作用不太关键。
