Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa
Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa
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DOI：
10.1006/jmbi.1998.1639
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发表时间：
1998-04-10
影响因子：
5.6
通讯作者：
Ruf, W
中科院分区：
文献类型：
--
作者：
Dickinson, CD;Shobe, J;Ruf, W
胰蛋白酶样丝氨酸蛋白酶凝血因子VIIa的催化活性受变构调节。在这项工作中，我们使用单克隆抗体作为探针来分析由酶原激活、辅因子组织因子（TF）结合和活性位点占用诱导的VII蛋白酶结构域的构象变化。三种单克隆抗体的表位是用蛋白酶结构域的57个丙氨酸替代突变体绘制的。其中两种抗体在活性位点裂口上方的基本簇中具有典型的“热点”表位，抗体与这些表位的结合不受酶原激活、TF结合或活性位点占用的影响。相比之下，VII/VIIa与与大分子底物外源位点重叠的扩展表位的单克隆抗体的结合动力学受到VIIa蛋白酶结构域的每个构象转变的影响。抗体亲和力的变化与酶原Till向TF的转变是一致的。VIIa复合体，游离酶VIIa作为中间体，保留了一些酶原样特征，导致其催化活性低。相反，活性位点占用导致的效果与酶原活化对抗体表位的影响有质的不同。这为这种变构调节的丝氨酸蛋白酶的活性位点、大分子底物识别区域和辅因子结合位点之间的构象相互依赖提供了新的见解。(C) 1998学术出版社有限公司
The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. Ln this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical "hot spot" epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen Till to the TF.VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This Provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease. (C) 1998 Academic Press Limited.