Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
St. Maurice, Martin
Second Advisor
Manogaran, Anita
Third Advisor
Reiter, Nicholas
Abstract
Pyruvate carboxylase (PC) produces oxaloacetate from pyruvate and bicarbonatein an ATP-dependent manner. The catalytic activity of PC places it at a pivotal intersection between catabolism and anabolism. Understanding the essential metabolic role of PC requires a more complete description of how its activity is regulated and how that regulation is manifested through changes in conformational dynamics. PC activity is regulated by the mutually exclusive binding of the allosteric activator, acetyl-CoA, and the allosteric inhibitor, L-aspartate. The binding site for acetyl- CoA has largely been identified, but the binding location for the acetyl moiety is unknown. Given that the acetyl moiety enhances the binding affinity drastically, a definition of the binding site will uncover new molecular insights regarding the allosteric mechanism. This work defines the binding site of the acetyl moiety at the biotin carboxylase dimer interface in Staphylococcus aureus PC and identifies essential residues Arg21, Lys46 and Glu418 to allosteric activation, and inhibition. Early studies on vertebrate PC reported enzyme-mediated hydrolysis of the acetyl moiety from acetyl-CoA. The complete definition of the acetyl moiety binding site offers new opportunities to understand enzyme-mediated hydrolysis. The current study confirms that microbial PC enzymes catalyze acetyl-CoA hydrolysis. Residues in the dimer interface protect against enzyme catalyzed hydrolysis of the acetyl moiety. This suggests that a secondary binding site in the carboxyltransferase domain is responsible for hydrolyzing acetyl-CoA. The regulatory mechanism that controls carrier domain translocation remains unknown. Using numerous biophysical tools, the current study demonstrates that the conformational dynamics of PC are altered by acetyl-CoA, increasing the rate of carrier domain translocation and coordinating carrier domain positioning in the tetramer. Acetyl- CoA does this by modulating the flexibility of the carboxyltransferase dimers at the corners of the tetramer. This work provides a molecular basis for both allosteric activation and inhibition and confirms that microbial PC enzymes catalyze acetyl-CoA hydrolysis. These studies provide insights into how allostery modulates the conformational dynamics of PC in a coordinated manner, lending greater insights into the metabolic role of PC at the crossroads of metabolism.