Structural Investigations of Productive Enzyme Complexes

描述（申请人？丝氨酸蛋白酶是一类酶， 在许多人类疾病中起着关键作用，包括癌症，囊性 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; 基于结构的药物设计已被临床成功充分证明， HIV蛋白酶抑制剂在抗击艾滋病中的作用。除了担任 作为药物靶标，丝氨酸蛋白酶也可用作药物靶点的有用模型。 阐明了酶用于催化的策略。尽管这一切 关于丝氨酸蛋白酶结构和机制已知， 可用于这些酶与其底物复合。这个缺失的部分 的信息源于一个技术难题;晶体结构提出了一个 分子图像随时间平均，而酶与它们的 靶通常反应太快而不能被标准晶体学捕获。 方法.技术的发展，包括强大的同步辐射 来源和冷冻同位素技术，现在已经使结构 测定其他几种酶与它们的天然复合物 印刷受体.该提议的第一个目的是获得晶体结构， 一种代表性的丝氨酸蛋白酶，枯草杆菌蛋白酶， 衬底第二个目标是使用为该结构开发的方法 测定第一枯草杆菌蛋白酶-底物复合物以获得结构 一系列突变的枯草杆菌蛋白酶复合物结构-反应性相关性 将绘制澄清的重要性，基板的方向在催化。 最后的目的是详细比较枯草杆菌蛋白酶底物的结构 与枯草杆菌蛋白酶-蛋白质抑制剂复合物的复合物，以确定 使一种络合物具有活性而另一种络合物不具有活性的结构特征。 由于活性中心几何构型和化学机理的保守性， 丝氨酸蛋白酶，结论得出的方向要求， 水解反应可推广到所有丝氨酸蛋白酶。采取 总之，成功完成这项工作所提供的信息 该提案将为未来的治疗设计提供一个改进的模板。 丝氨酸蛋白酶家族中的酶的抑制剂。

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.