Structure-function studies of glycosidases

我的实验室使用大肠杆菌中的一种酶，ß-半乳糖苷酶。这种酶有助于乳糖（牛奶中的糖）的分解。它与乳糖不耐症有关。这种酶是研究促进这类反应的酶的一个模型。这种酶也具有历史意义，因为它是遗传调控经典研究中使用的酶。本实验室的工作旨在确定这种酶是如何起作用的。酶的结构是已知的，我们知道活性位点上有哪些氨基酸（活性位点是酶上酶活性发生的区域）。活性位点的氨基酸一起催化反应。有些参与结合底物，有些参与稳定过渡态（反应中高度活跃的过渡中间形式），有些在催化反应的化学方面具有特定作用。此外，该酶还具有制造乳糖类似物（异丙乳糖）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），从而形成用于代谢乳糖的酶。我们正试图发现这种酶中每一种重要氨基酸的作用。我们通过一种叫做“位点特异性诱变”的方法来做到这一点，这种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能力。我们还将通过一种叫做“x射线晶体学”的方法来研究蛋白质的折叠方式。-半乳糖苷酶也需要镁才能正常工作，我们将进行研究以确定镁的作用。此外，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的研究。我们还计划从一种名为黑曲霉的生物中研究一种ß-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，因此研究它的性质是很重要的。

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.