Jin J, Chang J, Stafford D W, Straight D L
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Biochemistry. 2001 Sep 25;40(38):11405-10. doi: 10.1021/bi010990g.
We studied factor Xa activation of human factor VII in hopes of identifying factor VII residues, not adjacent to the cleavage site, involved in this interaction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with multiple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X activation, and the residues of MM4 are located at a hydrophobic patch of factor VII on the opposite side of the catalytic domain from those in MM3. Only H101A, Y179A, and MM4 were activated significantly more slowly than the wild type. Results of our kinetic analyses showed that the catalytic efficiency of factor Xa for activation of factor VII was 176- and 234-fold higher than that for H101A andY179A, respectively. All the mutants with measurable activity had affinities for tissue factor similar to those of the wild type. The activated hydrophobic patch residues, except N100A, which is adjacent to one of the catalytic residues, had normal activities toward both a small peptide substrate and factor X. The rest of the activated mutants (except D102Q with no activity) had reduced activities toward the small substrate (except R147A) and factor X. We conclude that factor VII activation by factor Xa and factor VIIa's catalytic interaction with factor X involve different regions in the catalytic domain, and residues H101 and Y179, part of an aromatic hydrophobic patch, are specifically involved in factor Xa activation of factor VII.
我们研究了人因子VII的因子Xa激活作用，以期确定参与这种相互作用的、不邻近裂解位点的因子VII残基。我们制备了8种具有单个突变的因子VII（N100A、H101A、D102Q、L144A、R147A、Y179A、D186A和F256A）以及2种具有多个突变的因子VII [MM3（L144A/R147A/D186A）和MM4（N100A/H101A/Y179A/F256A）]。MM3中的残基先前已被确定为影响因子X激活，而MM4的残基位于因子VII催化结构域另一侧的一个疏水区域，与MM3中的残基位置相对。只有H101A、Y179A和MM4的激活速度明显慢于野生型。我们的动力学分析结果表明，因子Xa激活因子VII的催化效率分别比H101A和Y179A高176倍和234倍。所有具有可测量活性的突变体对组织因子的亲和力与野生型相似。除了与一个催化残基相邻的N100A外，被激活的疏水区域残基对小肽底物和因子X均具有正常活性。其余被激活的突变体（除无活性的D102Q外）对小底物（R147A除外）和因子X的活性降低。我们得出结论，因子Xa对因子VII的激活以及因子VIIa与因子X的催化相互作用涉及催化结构域中的不同区域，并且作为芳香疏水区域一部分的残基H101和Y179特别参与因子Xa对因子VII的激活。
