Electrostatics and dynamics of glycosidases during reaction

Summary: The significance of these studies lies with both understanding fundamental aspects of enzymatic catalysis and providing the background necessary to engineer sugar-cleaving enzymes (glycosidases) with pH optima and stability profiles tailored for applications in biotechnology. Abstract: Cellulose and related polymeric carbohydrates, such as xylan, are the most abundant sources of carbon on earth. Therefore glycolytic enzymes, including cellulases and xylanases, have enormous potential roles in food and fuel production, environmentally-friendly pulp and paper processing, and synthesis of carbohydrate-based drugs. As such, these enzymes remain the subject of extensive research directed towards understanding the molecular mechanisms by which they specifically bind and hydrolyze their sugar substrates. The broad objectives of my research project are to define the roles played by electrostatic interactions and dynamics in establishing the structure and catalytic mechanism of glycosidases. As a model system, I will utilize the well defined Bacillus circulans endo-b-(1,4)-xylanase (Bcx). The main strategies to achieve these aims are mutational analyses to dissect the factors establishing the precise pKa values of the catalytic residues of Bcx and comparison of this model glycosidase with related xylanases from acidophilic/alkalophilic organisms. The primary methods are kinetic studies, combined with the use of NMR spectroscopy to measure the microscopic pKa values of ionizable groups in Bcx, as well as to characterize the motions of backbone and sidechain atoms along its reaction pathway.