Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding
Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding
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DOI：
10.1074/jbc.271.41.25332
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发表时间：
1996-10-11
影响因子：
4.8
通讯作者：
Mertens, K
中科院分区：
文献类型：
--
作者：
Lenting, PJ;Christophe, OD;Mertens, K
已知Ca 2+与因子IX的第一个表皮生长因子（EGF）样结构域的结合是生物活性所需的，但是Ca 2+促进因子IX功能的机制仍然不清楚。我们研究了缺乏Ca 2+与第一个EGF样结构域结合的重组因子IX突变体，这是由于Asp（64）被Glu、Lys或瓦尔取代，纯化的突变体（因子IX D 64 E、D 64 K和D 64 V）与血浆衍生的和重组野生型因子IX在许多金属离子依赖性功能参数方面进行比较。在Mg 2+存在下，活化的突变体在水解合成底物CH 3-SO 2-Leu-Gly-Arg-对硝基苯胺时与正常的因子IXa没有区别。用Ca ~（2+）替代Mg ~（2+）进一步刺激正常因子IXa的活性，但不刺激突变因子IXa的活性。在不依赖于因子Vm的因子X活化中，因子IXa D 64 K和D 64 E与正常因子IXa相比显示出降低的催化活性（表观k（cat）/K-m分别约为1、2和4 × 10（3）M（-1）s（-1）），在因子VIIIa存在下，正常和突变型因子IXa的因子X激活率在不同程度上受到因子VIIIa的刺激（分别约为700倍和200倍），表明Asp（64）替代影响与因子VIIIa的相互作用，这种可能性在使用包含因子VIII重链或轻链的因子IXa结合基序的合成肽的抑制研究中得到解决，而重链肽（Ser（558）-Gln（565））以相似的效率抑制正常和突变因子IXa对因子VIII依赖性因子X的激活，轻链肽（LyS（1804）-LyS（1818））抑制正常因子1Xa的效率是突变因子VIIla的2-3倍，这表明对因子VIIla的反应降低可能是由于突变因子IXa与因子VIII轻链的结合受损。在直接结合研究中进一步探讨了这一点。在Mg 2+存在下，正常和突变因子IXa与因子VIII轻链的结合相似。然而，在Ca 2+存在下，因子IXa突变体的效率低于正常因子IXa，这通过对因子VIII轻链的亲和力比正常因子IXa低4-5倍来说明。总的来说，我们的数据证明了许多因子IXa功能，包括酶活性和组装成因子IXa-因子VIIIa复合物，依赖于Ca 2+与因子IX的第一个EGF样结构域的结合。
Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear, We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp(64) by Glu, Lys, or Val, The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters, In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa, In factor Vm-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent k(cat)/K-m approximate to 1, 2, and 4 x 10(3) M(-1) s(-1), respectively), In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent (approximate to 700- and 200-fold, respectively), indicating that Asp(64) replacements affect the interaction with factor VIIIa, This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains, Whereas the heavy chain peptide (Ser(558)-Gln(565)) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys(1804)-Lys(1818)) inhibited normal factor 1Xa 2-3-fold more efficiently than did mutant factor VIIIa, This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies, In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain, Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.