Date of Award
Fall 2014
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
St. Maurice, Martin
Second Advisor
Anderson, James T.
Third Advisor
Eddinger, Thomas J.
Abstract
Pyruvate carboxylase (PC; E.C. 6.4.1.1), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated, and then translocate to the carboxyltransferase (CT) domain where the carboxyl group is transferred from biotin to pyruvate. Kinetic analyses of PC have suggested that the spatially distinct reactions, which occur in the active sites of the BC and CT domains, are well coordinated. To gain insights into the molecular events necessary for coordinating catalysis in the CT domain, structural and biochemical studies were performed. An in vitro enzyme assay was previously developed to measure the oxamate- induced decarboxylation of oxaloacetate in the CT domain. While this reaction has been widely utilized, the mechanism has not been fully established. Structural data reveals that oxamate is positioned in the active site of the CT domain in an identical manner to the substrate, pyruvate, and kinetic data demonstrates that the mechanism proceeds through a simple ping-pong bi bi mechanism. These results establish this assay as an ideal method to dissect the role of biotin in the isolated CT domain reaction. To investigate the molecular events necessary for facilitating biotin binding into the CT domain active site, structural investigations of the CT domain in the presence and absence of the substrate, product and intermediate analogs were conducted. These studies revealed a substrate-induced conformational change that is essential for catalysis. Site-directed mutations of two conformation-stabilizing residues result in a reduced rate of biotin-dependent reactions but have no effect on the rate of biotin-independent reactions, indicating that these residues are essential in facilitating biotin binding during catalysis. Furthermore, these structures reveal that CT domain ligands do not interact directly with the active site metal as previously hypothesized and that the substrate maintains its orientation throughout catalysis. Collectively, these studies reveal fundamental new insights into how substrate binding in the CT domain active site facilitates biotin binding during catalysis and contribute to a detailed description of how PC coordinates catalysis between two spatially distinct active sites.