Macromolecular Substrate Recognition in Blood Coagulation

凝血的特定蛋白分解激活步骤是由丝氨酸蛋白酶催化的，丝氨酸蛋白酶是 彼此同源，并与消化的原型丝氨酸蛋白酶同源。我们将使用凝血酶原 凝血酶原酶复合体作为特定大分子底物的范例所催化的激活 通过凝血复合体的识别和切割来研究酶功能的基础。我们的研究是 重点介绍了底物和酶之间的广泛相互作用在推动 凝血酶原的两个裂解部位上的凝血酶原酶。一系列方法将评估 伴随凝血酶原激活的酶原和蛋白水解酶之间的转换 将系留底物提供给酶并在以下过程中提供限速步骤 凝血酶的形成。新的相互作用物种的荧光衍生物将用于共振能量 转移研究以建立蛋白酶后底物呈递变化的物理相关性 底物-辅因子相互作用的形成及其在酶功能中的作用。约束性研究将确立 凝血酶原作为凝血酶原酶复合底物动力学行为的热力学基础。这些 方法将评估几何约束的作用，这些约束产生于外植体系链，允许酶 区分底物中的两个裂解位点。其他研究将评估网络终端机的作用 底物的结构域在参与外显子依赖的系留过程中。机械论的见解 将在规定条件下获得的结果推广到了解凝血酶原酶激活的功能 假设分裂途径和中间体产生的血小板和内皮细胞 取决于细胞类型，由细胞支持凝血酶原结合的能力决定。总的来说，我们的 策略基于新的概念，这些新概念来自于对底物识别的扩展理解 凝血酶原酶。拟议的方法将为以下方面提供新的线索： 与正常止血、疾病状态和治疗相关的酶功能的生物化学和生物学 靶向血栓和血管疾病的这种反应。

The specific proteolytic activation steps of blood coagulation are catalyzed by serine proteinases that are homologous to each other and to the archetypal serine proteinases <of digestion. We will use prothrombin activation catalyzed by the prothrombinase complex as a paradigm for specific macromolecular substrate recognition and cleavage by coagulation complexes to investigate the basis for enzymic function. Our studies are focused on the central role of extended interactions between substrate and enzyme in driving the action of prothrombinase on the two cleavage sites in prothrombin. A series of approaches will assess the role of the transition between zymogen and proteinase that accompanies the initial cleavage of prothrombin in regulating presentation of the tethered substrate to the enzyme and in providing a rate-limiting step in the process of thrombin formation. Novel fluorescent derivatives of the interacting species will be used in resonance energy transfer studies to establish physical correlates of changes in substrate presentation following proteinase formation and the role of substrate-cofactor interactions in enzymic function. Binding studies will establish the thermodynamic basis for the kinetic behavior of prothrombin as a compound substrate for prothrombinase. These approaches will assess the role of geometric constraints arising from exosite tethering that permit the enzyme to discriminate between the two cleavage sites in the substrate. Additional studies will assess the role of the Nterminal domain of the substrate in participating in the exosite-dependent tethering process. Mechanistic insights obtained under defined conditions will be extended to understand the function of prothrombinase on activated platelets and endothelial cells with the hypothesis that the cleavage pathway and intermediate produced is dependent on cell type, determined by the ability of the cells to support prothrombin binding. Overall, our strategies are based on novel concepts derived from an expanded understanding of substrate recognition by prothrombinase. The proposed approaches will shed new light on important yet poorly understood facets of the biochemistry and biology of enzyme function relevant to normal hemostasis, in disease states and for therapeutic targeting of this reaction in thrombotic and vascular disease.