Structural and mechanistic studies of Ser/Lys dyad proteases.

Ser/Lys 二元蛋白酶的结构和机制研究。

• 批准号: 250131-2012
• 负责人: Paetzel, Mark
• 金额: $ 2.91万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=547517
• 关键词: Structural; mechanistic; studies; Ser; Lys

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.

这项研究计划的目标是深入了解结构上无关的蛋白水解酶的结构和机制细节，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其底物中的肽键。有人认为，这些酶为催化赖氨酸的epsilon-氨基提供了一个微环境，使其能够去质子化，从而从催化的丝氨酸羟基中提取质子，激活它，使其对蛋白质底物的剪刀式羰基进行亲核攻击，从而作为一般碱基。最终，我们将使用X射线结晶学通过捕捉反应坐标的每个阶段来创建这些酶的作用的分子电影。发现酶在其催化循环的每个阶段之间的异同，并将结构数据与光谱和动力学数据相关联，将有助于我们理解Ser/Lys二联体机制的基本特征，并可能从总体上为酶催化的水解反应提供新的见解。许多Ser/Lys蛋白酶是细菌和病毒中的关键酶，因此我们将利用我们从研究中收集的信息来开发作为抗细菌和抗病毒化合物的抑制剂。在这项研究计划的拟议资助期内，我们将继续研究VP4蛋白酶的分子工作原理，它的功能是处理小核病毒中的多蛋白，小核病毒是一种感染鱼类和家禽的病毒，是导致水产养殖业和农业收入重大损失的疾病的原因。我们还将研究Traf蛋白水解酶，这是一种参与细菌接合菌毛组装的酶，有趣的是，它被认为同时具有蛋白酶和连接酶的活性，以产生环化蛋白产物。加拿大将从抗生素和抗病毒化合物的商业化中受益，这将导致对这些酶的研究。加拿大还将受益于高素质人员的培训。学生将在学术和工业领域都非常受欢迎的领域变得熟练和知识渊博。