Structual Correlates of Cofactor and Protease Function

非酶辅助因子Va(FVA)和丝氨酸蛋白酶因子Xa(FXA)是 这种酶复合体被称为凝血酶原酶，是已知的唯一的凝血酶原的生理激活剂。这个 这些活性物种的前体不能在任何程度上参与凝血酶原酶复合体， 这表明Fv到FVA和FX到FXA的蛋白水解性转换会导致适当的结构变化 赋予底物和酶结合能力。这项工作的广泛长期目标是 为了增加对原辅因子Fv和酶原Fx内的特定序列如何 有助于维持其相对不活跃的前体状态以及FVA中的序列如何调节相互作用 凝血酶原和凝血酶。我们将检验这一假设，即从B-空间限制的缓解- 结构域导致原癌基因切割后大分子结合位点的表达。我们还将 确定FV重链中的酸性序列是否提供对两种凝血酶的识别外位 和凝血酶原。我们假设这些酸性序列有助于原癌因子的激活，但不会帮助。 直接提高凝血酶原激活率。最后，我们将评估分子内激活是否 酶原到Protcase转换后的Fx导致功能结合位点在 蛋白酶域。我们假设Asp194通过内部盐桥的形成稳定下来 FX介导的高亲和力FVA和Na~*结合位点的表达而不是立体效应的去除 活化肽。综上所述，这些假设将通过生成一系列重组FX和 Fv变异体或血浆衍生的衍生物和这些蛋白质将使用平衡的组合进行评估 使用荧光和热力学技术的结合研究，蛋白质分解的动力学研究 反应和蛋白质结构研究。理解导致基因表达的分子事件 FVA和FXA的结构决定因素以及这些站点如何相互影响是开发的关键一步 针对血栓和血管疾病状态的高度特异性的药理药物。我们相信这一点 这一提议将有助于我们理解有关酶原蛋白和原辅酶因子的分子过程。 转变，这最终可能导致以意想不到的方式来调制属性FXA和FVA。

The nonenzymatic cofactor factor Va (FVa) and the serinc proteinase factor Xa (FXa) are essential components of the enzymatic complex termed prothrombinase, the only known physiological activator of prothrombin. The precursors of these active species cannot participate in the prothrombinase complex to any significant degree, indicating that thc proteolytic conversion of FV to FVa and FX to FXa results in appropriate structural changes that impart substrate and enzyme binding capability. The broad long-term objective of this work is directed towards increasing the understanding of how specific sequences within the procofactor FV and zymogen FX contribute to maintaining their relatively inactive precursor states and how sequences in FVa mediate interactions with prothrombin and thrombin. We will test the hypothesis that alleviation of steric constraints from the B- domain leads to the expression ofmacromolccular binding sites following procofactor cleavage. We will also determine whether acidic sequences within the heavy chain of FV provide a recognition exosite for both thrombin and prothrombin. We hypothesize that these acidic sequences facilitate procofactor activation, but do not assist directly in enhancing rates ofprothrombin activation. Finally, we will assess whether intrarnolecular activation of FX following the zymogen to protcase transition results in the expression of functional binding sites on the protease domain. We hypothesize that stabilization of Asp194 via internal salt bridge formation results in the expression of high affinity FVa and Na*-binding sites rather than removal of steric effects-mediated by the FX activation peptide. Taken together these hypotheses will be tested by generating a series of recombinant FX and FV variants or plasma-derived derivatives and these proteins will be evaluated using a combination of equilibrium binding studies employing fluorescence and thermodynamic techniques, kinetic studies of the proteolytic reactions, and protein structural studies. Understanding the molecular events leading to the expression of structural determinants on FVa and FXa and how these sites influence each other is a key step in the development of pharmacologic agents with high specificity aimed at thrombotic and vascular disease states. We believe this proposal will contribute to our understanding of the molecular processes involved in zymogcn and procofactor transitions, which may ultimately lead to unanticipated ways to modulate the properties FXa and FVa.