Structural and Biochemical Insights into Previously Uncharacterized Activity of Nucleotide-Dependent Phosphoenolpyruvate Carboxykinases

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) enzymes are central to glucose metabolism, with their main role in catalyzing one of the first steps of gluconeogenesis. In addition to its primary role, PEPCK is also involved, whether directly or indirectly, in glyceroneogenesis, amino acid metabolism, and lipogenesis. The PEPCK enzyme is responsible for the conversion of oxaloacetic acid to phosphoenolpyruvate using a triphosphate nucleotide as a phosphoryl donor. This dissertation focuses on human cytosolic PEPCK and Escherichia coli PEPCK as representative enzymes of the PEPCK family. The hcPEPCK enzyme is well characterized and extensively studied. Recent research suggests a new role for this enzyme, and potentially other GTP-dependent PEPCKs, as a protein kinase. hcPEPCK is thought to phosphorylate the ER protein INSIG, resulting in a cascade effect leading to increased lipogenesis. Expanding upon the information in the literature, phosphomimetic mutants were used to study the effects of phosphorylation on hcPEPCK, and its subsequent interactions with the INSIG protein. The ATP-dependent PEPCKs are less studied than their GTP counterparts, and some of the research in the literature is not up to date. Historically, the bulk of the research has involved GTP-dependent PEPCK. Using E. coli PEPCK as a proxy for the ATP-PEPCK class, a full kinetic and structural characterization was performed to further our understanding on these enzymes and compare differences and similarities between the classes. In addition, a new 31P-NMR assay was designed to address the hypothesis that ATP-dependent PEPCK can catalyze two additional reactions: the conversion of phosphoenolpyruvate to pyruvate (pyruvate kinase-like activity), and the nucleotide-dependent catalysis of phosphoglycolate.