Understanding enzyme-substrate recognition in catalysis and inhibition.

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Research in my lab is focused on the broad theme of using protein X-ray crystallography to understand molecular recognition in enzyme systems. By combining crystallography with enzymology and other biophysical techniques, we can characterize the roles of individual amino acids in ligand recognition. We are using state of the art facilities, including the Canadian Light Source, the Saskatchewan Structural Sciences Centre, and the Protein Characterization and Crystallization Facility located at the University of Saskatchewan. My lab has established its reputation by using structural studies to enhance our understanding of how enzymes recognize and bind their ligands. By examining these interactions at the atomic level, we can predict the effects of alterations on binding. We have the ultimate goal of using the information to develop compounds that can more effectively interact with enzymes and to use structural information to predict substrates for new, poorly understood enzymes. We have been using the thioredoxin system as a model system for studying protein-protein interactions. Our current research objectives are to probe the interactions between thioredoxin (Trx) and thioredoxin reductase (TrxR), to characterize the interactions that are important for binding and to examine the way these enzymes are compensating for temperature differences and other evolutionary pressures to maintain their interaction. We have chosen this system because the chemistry is very well characterized, the Trx system is ubiquitous and oxidative stress (in which Trx plays a regulatory role) is of great current scientific interest. By using crystallography and enzymology we are characterizing the interactions of the thioredoxin system from extremophiles to determine the recognition characteristics of this system. A full understanding of the interactions that determine the recognition and binding of the TrxR/Trx complex will offer insights into other protein-protein interactions. Our work also involves understanding how small molecules are specifically recognized by enzymes. In past grants, we have studied protein-ligand interactions for a variety enzymes, including inositol dehydrogenase (IDH), dihydrodipicolinate synthase (DHDPS) and UDP-galactopyranose mutase (UGM). We have determined the structures of these enzymes complexed with substrates and inhibitors in order to determine the modes of interactions and substrate recognition. We have utilized this information for to give us insights into how these diverse enzymes catalyze their reactions. Additionally, the information gained from our structural studies have aided us in identifying new sub-classes of enzyme families and to design novel enzyme inhibitors. In this grant, we propose to continue our work in using protein x-ray crystallography and enzymology to examine the interactions between novel enzymes and their ligands.