Catalytic Mechanisms and Biological Roles of Enzymes Involved in Energy Metabolism

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Structure dictates function. To understand how life works at the level of individual molecules, biochemists discern structures of biological molecules to learn how they function in a living cell. The field of structural biology is based on three experimental techniques: nuclear magnetic resonance spectroscopy, X-ray crystallography and cryoelectron microscopy. Each has limitations and advantages. Nuclear magnetic resonance spectroscopy looks at smaller macromolecules in solution where their dynamics can be detected. X-ray crystallography requires that the molecules be arranged in the ordered array of a crystal. Despite this limitation, a search of the Protein Data Bank shows that almost 90% of models are from X-ray crystallography. In part, this is because crystallographic data provide models of higher resolution, allowing details to be seen for even the larger macromolecules. Cryoelectron microscopy works best with large macromolecules whose different conformations can be investigated. Recent improvements in detectors allow single molecules to be visualized with the same amount of detail as from X-ray crystallography.******My research program focuses on investigating enzymes using the tools of structural biology to discover how the enzymes perform their biological roles. We study complex enzymes that have three or more substrates to understand how these multi-subunit enzymes with multiple active sites catalyze their reactions in living cells. Enzymes are a major target of drugs taken to improve the health of Canadians and understanding how they work guides the design of new drugs.******This proposal focuses on determining structures of two related multi-subunit enzymes, succinyl-CoA synthetase and ATP-citrate lyase. Both enzymes are involved in producing and using energy, but they have additional roles. Succinyl-CoA synthetase provides succinyl-CoA, which is used to make heme. ATP-citrate lyase provides acetyl-CoA to regulate the expression of genes. Our approach to understanding how enzymes work is to crystallize the enzyme with ligands. These ligands could be the molecules that react, products of the reaction or molecules that prevent the reaction. High-resolution models allow us to interpret how the enzyme catalyzes the reaction. When we struggle to grow good crystals, we use small angle X-ray scattering and cryoelectron microscopy.******Our research is important for understanding how these enzymes work and why succinyl-CoA synthetase is necessary to prevent X-linked sideroblastic anemia. Succinyl-CoA synthetase is not a drug target but ATP-citrate lyase is. Since the two enzymes are related, our insight into how each works will help the pharmaceutical industry design new drugs that inhibit ATP-citrate lyase to treat cancer or obesity.