Structural Investigations of Productive Enzyme Complexes

描述(申请人？S摘要)：丝氨酸蛋白酶是一类酶 在许多人类疾病中发挥关键作用，包括癌症、囊肿性疾病 纤维化、类风湿性关节炎、肺气肿、血栓栓塞性疾病、丙型肝炎、 和巨细胞病毒。高特异性丝氨酸蛋白酶抑制剂的设计 是一种很有前途的疾病治疗方法；潜在的 基于结构的药物设计已被临床成功充分证明。 艾滋病毒蛋白水解酶抑制剂在抗击艾滋病方面的应用。除了担任 药物靶点，丝氨酸蛋白酶也是一种有用的模型 阐明酶用于催化的策略。尽管如此， 已知丝氨酸蛋白酶的结构和机制，目前还没有解决的结构 可用于这些酶与其底物的络合。这块遗失的碎片 的信息源于技术难题；晶体结构呈现出 分子图像随着时间的推移而平均，而酶与它们的 靶通常反应太快，不能被标准的结晶学捕获 方法：研究方法。技术发展，包括强大的同步辐射 来源和冰晶技术，现在使结构成为可能 其他几种酶与其天然配位酶的测定 底物。这个提议的第一个目标是获得一种晶体结构，用于 一种具有代表性的丝氨酸蛋白酶，枯草杆菌酶，与特定的 底物。第二个目标是使用为结构开发的方法 确定第一个获得结构的枯草杆菌菌素-底物复合体 一系列突变的枯草杆菌复合体。结构-反应性相关性 将阐明底物取向在催化中的重要性。 最终目的是详细比较枯草杆菌菌素底物的结构。 与枯草杆菌蛋白抑制剂复合体的复合体，以确定 使一个复合体具有反应性而使另一个复合体无反应性的结构特征。 由于活性中心几何结构和化学机理的守恒性 丝氨酸蛋白酶，关于丝氨酸蛋白酶取向要求的结论 水解反应将适用于所有丝氨酸蛋白酶。已被占用 总之，成功完成这项工作所提供的信息 提案将为未来的治疗性设计提供一个改进的模板 丝氨酸蛋白酶家族中的酶抑制剂。

DESCRIPTION (applicant?s abstract): Serine proteases are a class of enzymes that play critical roles in many human diseases, including cancer, cystic fibrosis, rheumatoid arthritis, emphysema, thromboembolic disease, hepatitis C, and cytomegalovirus. The design of highly specific serine protease inhibitors is a promising therapeutic approach for disease treatment; the potential of structure-based drug design has been amply demonstrated by the clinical success of HIV protease inhibitors in combating AIDS. In addition to serving as pharmaceutical targets, serine proteases also serve as a useful model for elucidating the strategies that enzymes use for catalysis. Despite all that is known about serine protease structure and mechanism, no solved structures are available for these enzymes complexed with their substrates. This missing piece of information stems from a technical difficulty; crystal structures present a molecular picture averaged over time, while enzymes complexed with their targets usually react too quickly to be captured by standard crystallographic methods. Developments in technology, including powerful synchrotron radiation sources and cryocrystallographic techniques, now have enabled structure determination for several other enzymes complexed with their natural substrates. The first aim of this proposal is to obtain a crystal structure for a representative serine protease, subtilisin, complexed with a specific substrate. The second aim is to use the methodology developed for the structure determination of the first subtilisin-substrate complex to obtain structures for a series of mutant subtilisin complexes. Structure-reactivity correlations will be drawn to clarify the importance of substrate orientation in catalysis. The final aim is to compare in detail the structure of a subtilisin-substrate complex with that of a subtilisin-protein inhibitor complex, to determine the structural features that make one complex reactive and the other unreactive. Because of the conservation of active site geometry and chemical mechanism in serine proteases, conclusions drawn about the orientation requirements for the hydrolysis reaction will be generalizable to all serine proteases. Taken together, the information provided by the successful completion of this proposal will provide an improved template for the future design of therapeutic inhibitors of enzymes in the serine protease family.