Structure-function studies of glycosidases

糖苷酶的结构功能研究

• 批准号: 4691-2009
• 负责人: Huber, Reuben
• 金额: $ 1.82万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2013
• 资助国家: 加拿大
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=520727
• 关键词: Structure; function; studies; glycosidases

My laboratory works with the enzyme, ß-galactosidase, from Escherichia coli. This enzyme facilitates the breakdown of lactose (milk sugar). It is involved in lactose intolerance. The enzyme is a model for studies of enzymes that facilitate these types of reactions. The enzyme is also of historical significance as it was the enzyme used in the classical study of genetic regulation. The work in this laboratory is designed to determine how the enzyme functions. The structure of the enzyme is known and we know what amino acids are at the active site (the active site is the area on the enzyme at which the enzyme activity takes place). Amino acids at the active site work together to catalyze the reaction. Some are involved in binding the substrate, some in stabilizing the transition state (the highly active transient intermediate form in the reaction) and some have specific roles in chemical aspects of the catalytic reaction. In addition, the enzyme also has a function in making a lactose analog (allolactose), an important transient product that induces the genetic machinery (called the lac operon) that causes the formation of enzymes designed to metabolism lactose. We are attempting to discover the role of every important amino acid in this enzyme. We are doing this by a method called "site specific mutagenesis" that allows us to substitute specific amino acids. If an amino acid is important, the substitution should affect how well the enzyme binds reactants and forms products. We will also study the way that the protein is folded by a method called "X-ray crystallography". ß-Galactosidase also requires magnesium to function properly and we will carry out studies to determine the role of the magnesium. In addition there is an important loop region on the enzyme that is needed for full activity. It will be studied in detail. We are also planning to study a ß-glucosidase from an organism named Aspergillus niger - mainly we will be determining its structure (by X-ray crystallography). It is involved in cellulose breakdown and thus study of its properties is important.

我的实验室研究的是大肠杆菌中的β-半乳糖苷酶。这种酶促进乳糖（乳糖）的分解。它与乳糖不耐症有关。这种酶是研究促进这些类型反应的酶的模型。这种酶也具有历史意义，因为它是遗传调节经典研究中使用的酶。这个实验室的工作旨在确定酶是如何起作用的。酶的结构是已知的，我们知道活性位点上的氨基酸是什么（活性位点是酶上发生酶活性的区域）。活性位点上的氨基酸共同催化反应。有些参与结合底物，有些稳定过渡态（反应中高度活跃的瞬态中间体形式），有些在催化反应的化学方面具有特定的作用。此外，该酶还具有制造乳糖类似物（异乳糖）的功能，这是一种重要的瞬时产物，可诱导遗传机制（称为乳糖操纵子），从而形成旨在代谢乳糖的酶。我们正试图发现这种酶中每一个重要氨基酸的作用。我们正在通过一种称为“位点特异性诱变”的方法来实现这一点，该方法允许我们替换特定的氨基酸。如果一个氨基酸是重要的，取代应该影响酶结合反应物和形成产物的程度。我们还将通过一种称为“X射线晶体学”的方法研究蛋白质折叠的方式。β-半乳糖苷酶也需要镁才能正常发挥作用，我们将进行研究以确定镁的作用。此外，在酶上有一个重要的环区域，这是完全活性所必需的。将对此进行详细研究。我们还计划研究一种来自尼日尔曲霉的β-葡萄糖苷酶，主要是确定它的结构（通过X射线晶体学）。它参与纤维素的分解，因此对其性质的研究是重要的。