The Prothrombinase Complex: A Model of an Enzyme-Cofactor Complex

描述（由申请方提供）：通过两个级联的微妙平衡维持止血，导致凝块的形成（凝血）和溶解（纤维蛋白溶解）。凝血级联反应通过形成三个连续的酶-辅因子复合物来传播，这迅速导致大量凝血酶的产生。凝血酶原酶复合物，在酶因子Xa和辅因子Va之间的非共价缔合之间形成，用作凝血酶的最终产生者。如果没有这种复合物，就不能在足够的时间内形成生理相关水平的凝血酶，从而在凝血发生之前形成凝块。虽然凝血酶原酶复合物的动力学已经非常详细，但因子Xa和Va之间导致酶活性大幅调节的缔合机制还没有。分析酶-辅因子界面和复合物结构的残基对于理解相互作用的机制是必要的。该提议的目的是：（1）确定形成完全活性凝血酶原酶复合物所需的辅因子内以及辅因子Va和酶因子Xa之间的非共价相互作用的分子机制;和（2）确定凝血酶原酶复合物的X射线晶体结构。最近的晶体结构的失活因子Va已确定潜在的区域的辅因子稳定，并导致几个计算机模型提出的区域的相互作用，在酶-辅因子界面。将分析参与辅因子稳定和酶-辅因子相互作用的相关残基突变对凝血酶原酶活性和调节的影响。酶和辅因子上的协调突变将识别在界面处发现的非共价相互作用和在凝血酶生成中的作用。凝血酶原酶复合物的X射线结构最有可能用于确认界面处的残基以及鉴定其他残基和相互作用。对所涉及的蛋白质和复合物的生物化学和结构的理解将使靶向药物设计能够帮助维持止血。凝血级联的药理学干预和调节是临床必需的，超过40%的美国人口患有某种形式的心血管疾病。

DESCRIPTION (provided by applicant): Hemostasis is maintained through a delicate balance of two cascades leading to the formation (coagulation) and dissolution (fibrinolysis) of a clot. The coagulation cascade is propagated through formation of three successive enzyme-cofactor complexes that rapidly leads to generation of large quantities of thrombin. The prothrombinase complex, formed between a noncovalent association between the enzyme factor Xa and cofactor Va, serves as the final generator of thrombin. Without this complex physiologically relevant levels of thrombin cannot be formed in a sufficient amount of time to form a clot before hemorrhaging occurs. While the kinetics of the prothrombinase complex have been greatly detailed, the mechanism of association between factors Xa and Va that leads to large modulations in enzymatic activity is not. Analysis of the residues at the enzyme-cofactor interface and complex structure is necessary to understand the mechanism of interaction. The aims of this proposal are to: (1) determine the molecular mechanism of the noncovalent interactions, both within the cofactor as well as between the cofactor Va and enzyme factor Xa, required for formation of a fully active prothrombinase complex; and (2) determine the x-ray crystallographic structure of the prothrombinase complex. A recent crystal structure of inactivated factor Va has identified potential regions of cofactor stabilization and led to several computer models proposing regions of interaction at the enzyme-cofactor interface. Mutations of implicated residues involved in cofactor stabilization and enzyme- cofactor interactions will be analyzed for their effect on prothrombinase activity and regulation. Coordinated mutations on both enzyme and cofactor will identify the noncovalent interactions found at the interface and role in thrombin generation. The x-ray structure of the prothrombinase complex has the most potential for confirming residues at the interface as well as identifying additional residues and interactions. A biochemical and structural understanding of involved proteins and complexes will enable targeted drug design to assist in maintaining hemostasis. Pharmacological intervention and regulation of the coagulation cascade is a clinical necessity with more than 40% of the U.S. population suffering from some form of cardiovascular disease.