治疗酶是一类重要的生物药物，目前许多具有潜在治疗价值的天然酶分子因催化活性低、稳定性差，导致成药性低。脯氨酰内肽酶（SC PEP）是治疗发病率最高的人类遗传病之一的乳糜泻的候选药物，但其催化活性低、口服给药易降解，采用已有的酶分子设计方法仍未能显著提高SC PEP的催化活性与稳定性，难以成药，其原因在于SC PEP催化反应路径和酶分子结构的动态变化等关键机理至今尚未阐明。为此本项目深入模拟SC PEP催化反应过程，确定SC PEP催化反应路径，揭示SC PEP催化活性与关键氨基酸残基的关系，系统阐明其催化机理和稳定性的机理，根据催化机理设计与发现用于治疗乳糜泻的具有高催化活性和稳定性的口服SC PEP新药候选分子，为合理化设计与发现高效稳定的酶类新药提供科学依据和新策略新方法。

Enzymes drugs are an important class of biopharmaceutics. Now many natural enzymes with potential therapeutic value have low druggability, because of their low catalytic activity and poor stability. Prolyl endopeptidase form Sphingomonas capsulata (SC PEP) is a drug candidate for celiac sprue, which is one of the human genetic diseases with the highest incidence, but SC PEP has low catalytic activity and is easily biodegradable. Unfortunately, enzymes designed by using the conventional design methods have the problems of low catalytic activities and stability, and the enzyme therapy development is limited by the commonly recognized “stability-function tradeoff” theory. Therefore, the conventional design methods could not significantly increase catalytic activity and stability of SC PEP. There is no research revealing the enzymatic reaction pathway of SC PEP and the dynamic changes of enzyme structure. Therefore the catalytic mechanism of SC PEP is still not accurately clarified. Nevertheless, our recently developed unique computational enzyme modeling strategies and methods (e.g. the structure and transition-state based modeling) are promising in solving these challenges. Taking advantage of these recently developed computational enzyme modeling and design strategies and methods, in this project, we will reveal the catalytic mechanism of SC PEP, rationally redesign, discover, and develop prolyl endopeptidase (PEP), aiming to develop an engineered PEP with significantly improved catalytic activities against immunodominant gluten peptides and stability at low pH suitable for use as a novel oral enzyme therapy. To achieve the goal, we will first computationally uncover the detailed mechanism for PEP-catalyzed hydrolysis of immunodominant gluten peptides, followed by structure and mechanism based design of new mutants that are stable at low pH and potentially have improved catalytic activities against immunodominant gluten peptides. The computationally designed mutants will be evaluated in vitro for their actual catalytic activities, selectivity, and stability that in turn will be used to refine the computational design protocol and to make the next round of computational design more reliable. Thus, the iterative computational-experimental studies should eventually lead to reveal the catalytic mechanism of SC PEP, and to discovery of an PEP form with the desired catalytic activities, selectivity, and stability. Completion of this project will result in discovery of highly efficient prolyl endopeptidases metabolizing immunodominant gluten peptides suitable for further development as novel oral enzyme therapies to treat gluten-related diseases, and will demonstrate a generally-applicable protocol to rationally design and discover highly efficient therapeutic enzymes.