Structural and mechanistic studies of Ser/Lys dyad proteases

拟议的研究计划的目标是调查两个不相关的蛋白水解酶家族(一个细菌和一个病毒)的结构和机制细节，它们进化出相同的不寻常的催化机制。这些蛋白酶；细菌信号肽酶和病毒VP4蛋白水解酶在它们的水解反应中都利用了必需的赖氨酸和丝氨酸。去质子化的赖氨酸被认为是普通的碱基，它从丝氨酸羟基中提取质子，从而激活它，对底物的剪切式羰基进行亲核攻击。信号肽酶是一种基本的细菌酶，其功能是从细胞膜释放分泌的蛋白质，许多制药公司正在研究它作为新的抗菌靶点。VP4蛋白酶参与了对禽类病毒外壳蛋白的蛋白水解性加工，这种传染病每年给养鱼业和农业造成数十亿美元的收入损失。我们将使用分子生物学、蛋白质化学、X射线结晶学、酶动力学和核磁共振来研究这两个酶家族的结构和机制的异同，以便更详细地了解Ser/Lys二联体机制是如何工作的，以及酶是如何工作的。加拿大将从这些酶的基于结构的合理抗菌和抗病毒药物设计所产生的商业化药物中受益。加拿大也将从我们实验室高素质人员的培训中受益。学生和博士后研究员正在成为拥有分子生物学、蛋白质化学和X射线结晶学技能的结构生物学家(在学术界和工业界都很受欢迎)。

The objectives of the proposed research program is to investigate the structural and mechanistic details of two unrelated families of proteolytic enzymes (one bacterial and one viral) that have evolved the same unusual catalytic mechanism. These proteases; bacterial signal peptidase and viral VP4 protease both utilize an essential lysine and serine in their hydrolytic reactions. The deprotonated lysine is thought to serve as the general-base by abstracting the proton from the serine hydroxyl thereby activating it for nucleophilic attack on the scissile carbonyl of the substrate.Signal peptidase is an essential bacterial enzyme that functions to release secreted proteins from the cell membrane and is being investigated by a number of pharmaceutical companies as a novel antibacterial target.  VP4 protease is involved in the proteolytic processing of viral coat proteins in birnavirus, the infectious agent that costs the fish farming and agricultural industry billions of dollars of lost revenue each year. We will use; molecular biology, protein chemistry, X-ray crystallography, enzyme kinetics, and Nucleur Magnetic Resonance to investigate the structural and mechanistic similarities and difference in these two enzymes families in order to provide a more detailed understanding of how the Ser/Lys dyad mechanism works, and how enzymes work in general.Canada will benefit socially and financially from the commercialized drugs that result from the structure-based rational antibacterial and antiviral drug design efforts on these enzymes.  Canada will also benefit from the training of highly qualifies personnel in our lab. Students and post-doctoral fellows are becoming structural biologist (highly sought after in both academics and industry) with skills in molecular biology, protein chemistry and X-ray crystallography.