Structure-function studies of glycosidases

点击翻译按钮获取中文摘要

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.