Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity
Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity
复制标题
DOI：
10.1074/jbc.275.1.229
复制
发表时间：
2000-01-07
影响因子：
4.8
通讯作者：
Mertens, K
中科院分区：
文献类型：
--
作者：
Celie, PHN;Lenting, PJ;Mertens, K
活化因子IX的三维结构包括两个表皮生长因子（EGF）样结构域之间的多个接触。其中之一是Glu（78）和Arg（94）之间的盐桥，其对于因子Ma与其辅因子VIII的结合和因子VIII依赖性因子X活化是必需的（Christophe，O. D、伦廷，P. J.，Kolkman，J. A. Brownlee，G. G.，Mertens，K.（1998）J.Biol.Chem.273，222-227）。我们现在讨论了EGF样结构域之间界面处的假定疏水接触。构建了重组因子IX嵌合体，其中疏水区域Phe（75）-Phe（77）和Lys（106）-瓦尔（108）被因子X和因子VII的相应位点取代。活化的因子IX/因子X嵌合体在因子Ma酶活性方面与正常的因子Ma不可区分。相比之下，在存在因子VIII的情况下，因子IXa（75-77）/因子VII显示出与2倍增加的因子X激活相似的作用，这表明残基75-77有助于辅因子依赖性因子X激活。因子IX 106 -108/因子VII对因子X的激活在因子VIII存在和不存在的情况下均强烈降低。活性可以恢复，同时取代的疏水性位点在两个EGF样结构域因子VII残基。这些数据表明，因子Ma酶活性需要两个EGF样结构域之间的疏水接触。
The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor Ma to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A. Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains, Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII, Activated factor IX/factor X chimeras were indistinguishable from normal factor Ma with respect to factor Ma enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed similar to 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX106-108/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor Ma enzymatic activity requires hydrophobic contact between the two EGF-like domains.