UC Berkeley

Phosphoenolpyruvate carboxykinase (PCK) is a key metabolic enzyme responsible for catalyzing the first committed step of gluconeogenesis, the conversion of oxaloacetate to phosphoenolpyruvate and carbon dioxide, in all living organisms. PCK has been implicated in diabetes mellitus along with tumor growth under glucose-depleted conditions. Despite its critical role in metabolism and health, certain key aspects of this enzyme remain uncharacterized. The work in this dissertation focuses on understanding the relationship of structures, conformations, and ligand specificity for the E. coli isozyme of PCK through structural X-ray methods.

Because PCK directly utilizes CO2, the CO2 binding pocket can be engineered to accommodate different substrates to enzymatically form carbon-carbon bonds. Chapter 2 details results for six crystal structures with mutations in or near the CO2 binding site that lead to binding of the nonnative ligands thiosulfate and methanesulfonate as defined by macromolecular crystallography. Even though these ligands both contain a sulfonate moiety, mutants are found to bind only one or the other, proving to dissect aspects of ligand recognition. The orientation of these ligands also are altered by the hydrogen bonding network present in the pocket of interest. Through computational simulations, these phenomena are rationalized through the displacement of loosely bound water molecules in the binding pocket.

Conformational changes are crucial to the catalytic cycle of many enzymes, and PCK is no exception. Chapter 3 discusses my efforts to better understand how PCK undergoes conformational changes in different circumstances. First, insertion mutants in new intermediate crystallographic conformations were characterized and a metric to measure the closedness of the enzyme was developed. Crystallographic models provide accurate atomic information but are not comprehensive for conformational states, so I go beyond crystallographic models to examine the solution state conformations of PCK. This more accurately reflects biological conditions when compared to models obtained from crystal structures because the solution state is free of influences such as crystal packing and the experimental conditions are more realistic relative to those used to grow crystals. To examine solution state conformations, small angle X-ray scattering (SAXS) data were collected on EcPCK and 10 of its mutants under various reaction conditions resulting in 33 experimental profiles. Interestingly, the apo state in solution did not correspond to its crystal structure; instead, it adopted a more closed conformation. Furthermore, the ATP-bound state of EcPCK has only been found in the cap-open state, where the cap is a flexible loop that closes over the active site cleft, while the SAXS data supports a closed-cap conformation. Given these different results in solution and the value of comparing them comprehensively to crystal structures, I present a novel way of visualizing SAXS data in high-throughput, using PCK as the model system.

Together, the results and concepts from this work provide insight into a key region of the EcPCK active site cleft and to the behavior of the system as a whole in solution. These data and ideas further our understanding of this enzyme, and to pave the way toward characterizing enzymes in their solution states as they undergo changes during catalysis.