Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191.
Intrinsic pathway activation of factor X and its activation peptide-deficient derivative, factor Xdes-143-191.
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DOI：
10.1016/s0021-9258(18)42587-2
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发表时间：
1992-04
期刊：
影响因子：
--
通讯作者：
E. J. Duffy;P. Lollar
中科院分区：
文献类型：
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作者：
E. J. Duffy;P. Lollar
利用一种新型的fX衍生物研究了激活肽在决定内在途径因子X（fX）激活的底物特异性中的作用，其中52个残基的激活肽的NH 2末端被来自蛇的酶解去除了49个残基。红口蝮蛇。被命名为fXdes-143-191的修饰蛋白是无活性的，但被由因子IXa β、凝血酶活化因子VIII（fVIIIaIIa）和磷脂囊泡组成的内在fX活化复合物（内在fX酶）或被来自拉塞尔蝰蛇毒液的fX凝血蛋白（RVV-XCP）活化为α-fXa。RVV-XCP激活fX的Km和kcat均大于fXdes-143-191，导致催化效率（kcat/Km）的差异小于2倍，表明fXdes-143-191与RVV-XCP的非生产性结合。内源性fXase对每种底物的活化显示，fX的kcat是fXdes-143-191的100倍（分别为16和0.16 s-1），尽管Km没有可检测到的差异（分别为60和80 nM）。在不存在fVIIIaIIa的情况下，fIXa β/磷脂的激活也揭示了kcat的差异，但没有Km的差异，但kcat的差异较小（fX和fXdes-143-191的kcat分别为0.007和0.002 s-1，Km分别为220和170 nM）。产物对时间曲线的分析表明，fVIIIaIIa促进在fX活化过程中形成酰基-酶中间体。我们的结论是，激活肽在酰基酶形成过程中起着关键作用，这是最明显的fVIIIaIIa的存在下。Km差异的情况下表明，残基NH 2-末端P3不有助于酶-底物复合物的初始形成。
The role of the activation peptide in determining the substrate specificity of intrinsic pathway factor X (fX) activation was studied by using a novel derivative of fX in which 49 residues were removed enzymatically from the NH2 terminus of the 52-residue activation peptide by an enzyme from the venom of the snake Agkistrodon rhodostoma. The modified protein, designated fXdes-143-191, is inactive but is activated to alpha-fXa by either the intrinsic fX activation complex (intrinsic fXase) composed of factor IXa beta, thrombin-activated factor VIII (fVIIIaIIa), and phospholipid vesicles or by the fX coagulant protein from Russell's viper venom (RVV-XCP). Both the Km and kcat for the activation of fX by RVV-XCP were greater than for fXdes-143-191, resulting in less than a 2-fold difference in the catalytic efficiency (kcat/Km) suggestive of nonproductive binding of fXdes-143-191 to RVV-XCP. The activation of each substrate by intrinsic fXase revealed that the kcat was 100-fold greater for fX than fXdes-143-191 (16 and 0.16 s-1, respectively), although there was no detectable difference in Km (60 and 80 nM, respectively). Activations by fIXa beta/phospholipid in the absence of fVIIIaIIa also revealed a difference in kcat but not Km, but the difference in kcat was smaller (kcat of 0.007 and 0.002 s-1 and Km of 220 and 170 nM for fX and fXdes-143-191, respectively). Analysis of product versus time curves demonstrated that fVIIIaIIa promotes formation of the actyl-enzyme intermediate during fX activation. We conclude that the activation peptide plays a critical role during acyl-enzyme formation that is most pronounced in the presence of fVIIIaIIa. The absence of Km differences suggests that residues NH2-terminal to P3 do not contribute to the initial formation of the enzyme-substrate complex.