Structural and mechanistic studies of Ser/Lys dyad proteases.

这项研究计划的目标是深入了解结构上不相关的蛋白水解酶的结构和机理细节，这些酶已经进化到利用不寻常的Ser/Lys催化二联体机制来水解底物中的肽键。有人提出，这些酶提供了一个微环境的ε-氨基的催化赖氨酸，使它能够成为去质子化，从而作为一般的碱，从催化丝氨酸羟基提取质子，激活它的亲核攻击的易裂羰基的蛋白质底物。最终，我们将使用X射线晶体学通过捕获反应坐标的每个阶段来创建这些酶的分子电影。发现酶在其催化循环的每个阶段之间的相似性和差异，并将结构数据与光谱和动力学数据相关联，将使我们能够理解Ser/Lys二联体机制的基本特征，并可能为酶催化水解反应提供新的见解。许多丝氨酸/赖氨酸蛋白酶是细菌和病毒中的必需酶，因此我们将利用我们从研究中收集的信息来开发作为抗菌和抗病毒化合物的抑制剂。在本研究计划的拟议资助期间，我们将继续研究VP 4蛋白酶的分子工作机制，该蛋白酶的功能是处理birnaviruses中的多蛋白，birnaviruses是一种感染鱼类和家禽的病毒，是导致水产养殖和农业收入显著损失的疾病的原因。我们还将研究TraF蛋白酶，一种参与细菌接合皮利组装的酶，有趣的是，它被认为具有蛋白酶和连接酶的活性，可以产生环化的蛋白质产物。加拿大将从抗生素和抗病毒化合物的商业化中获得社会和经济利益，这些化合物是对这些酶的研究的结果。加拿大还将受益于高素质人员的培训。学生将成为在学术和工业部门都非常抢手的领域的技能和知识。

The goal of this research program is to gain insight into the structural and mechanistic details of architecturally unrelated proteolytic enzymes that have evolved to utilize the unusual Ser/Lys catalytic dyad mechanism for the hydrolysis of the peptide bond in their substrates. It is proposed that these enzymes provides a microenvironment for the epsilon-amino group of the catalytic lysine such that it is able to become deprotonated and thereby serve as the general-base by abstracting the proton from the catalytic serine hydroxyl, activating it for nucleophilic attack on the scissile carbonyl of the protein substrate. Ultimately we will use X-ray crystallography to create a molecular movie of these enzymes in action by trapping each stage of the reaction coordinate. Discovering the similarities and differences between the enzymes at each stage of their catalytic cycle and correlate the structural data with spectroscopic and kinetic data will allow us to understand the essential features of the Ser/Lys dyad mechanism and possibly provide new insights into enzyme catalyzed hydrolysis reactions in general. Many of the Ser/Lys proteases are essential enzymes in bacteria and viruses, therefore we will to use the information we glean from our investigations to develop inhibitors that work as anti-bacterial and anti-viral compounds. In the proposed funding period of this research program we are continuing our investigation into the molecular workings of VP4 proteases which functions to process the polyprotein in birnaviruses, a virus that infects fish and poultry and is the cause of diseases that result in significant lost revenues in the both the aquaculture and agriculture industries. We will also be investigating TraF protease, an enzyme involved in bacterial conjugative pili assembly and interestingly is proposed to have both protease and ligase activity to produce a cyclized protein product. Canada will benefit socially and financially from the commercialization of antibiotic and antiviral compounds that result for the investigation in these enzymes. Canada will also benefit from the training of highly qualifies personnel. Students will become skilled and knowledgeable in fields that are highly sought after in both academic and industrial sectors.