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: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=419943
• 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.

我的实验室使用的是来自大肠杆菌的半乳糖苷酶。这种酶有助于乳糖(乳糖)的分解。它与乳糖不耐受有关。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，因为它是经典的遗传调控研究中使用的酶。这个实验室的工作是为了确定这种酶的功能。酶的结构是已知的，我们知道活性部位是什么氨基酸(活性部位是酶上发生酶活性的区域)。活性部位的氨基酸共同作用催化反应。一些参与与底物结合，一些参与稳定过渡态(反应中的高活性瞬时中间形式)，还有一些在催化反应的化学方面具有特定的作用。此外，这种酶还具有制造乳糖类似物(别乳糖)的功能，这是一种重要的瞬时产物，它诱导遗传机制(称为乳胶操纵子)，导致形成旨在新陈代谢乳糖的酶。我们正试图发现这种酶中每一种重要氨基酸的作用。我们是通过一种叫做“定点突变”的方法来完成这项工作的，这种方法可以让我们用特定的氨基酸来替代。如果一种氨基酸是重要的，取代应该影响酶结合反应物和形成产物的好坏。我们还将研究蛋白质折叠的方式，这种方法被称为“X射线结晶学”。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，这是充分发挥活性所必需的。我们将对此进行详细研究。我们还计划研究一种名为黑曲霉的生物的β-葡萄糖苷酶-主要是我们将确定其结构(通过X射线结晶学)。它与纤维素的分解有关，因此对其性质的研究很重要。