Date of Award

Fall 2018

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Schläppi, Michael R.

Second Advisor

Maurice, Martin St.

Third Advisor

Silvaggi, Nicholas R.


Pyruvate carboxylase (PC; E.C. is a multifunctional, biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate. The overall reaction is accomplished by the coupling of two half reactions occurring at two spatially distinct catalytic domains by the translocation of a carrier domain, resulting in a net transfer of CO2 from bicarbonate to pyruvate. PC activity is regulated by multiple allosteric effectors with acetyl CoA serving as an activator in most species and L-aspartate serving as an inhibitor for microbial PC. The kinetic characterization of PC from different species have revealed that PC homologs are subject to divergent degrees of allosteric regulation, yet the mechanism controlling the allosteric response still remains unknown. Rhizobium etli (RePC) is allosterically activated by acetyl CoA, while Aspergillus nidulans (AnPC) is unique in not being activated by acetyl CoA. The divergent regulatory properties of these two PC homologs were investigated in order to uncover the fundamental mechanism underlying the allosteric regulation of pyruvate carboxylase. The lack of sensitivity to acetyl CoA activation was determined to be an intrinsic property of the BC domain rather than a consequence of different acetyl CoA binding sites. Further characterization of AnPC compared to RePC revealed that AnPC is a highly stable tetramer that remains in a permanently activated state. A more ordered transition state and coupled half reactions contribute to its high intrinsic catalytic rate as well as the insensitivity to the activation by acetyl CoA. Investigations on the translocation of the carrier domain revealed that multiple translocation pathways are adopted by the carrier domain in PC during catalysis. Interestingly, an intermolecular translocation pathway was promoted upon binding with acetyl CoA in RePC, which likely contributes to the enhanced coupling efficiency as well as the elevated catalytic rate observed in RePC. Finally, studies on the allosteric inhibition of PC by L-aspartate revealed that the antagonistic effects of acetyl CoA and L-aspartate to the catalysis of PC result from their competitive binding at a partially overlapping binding site. Collectively, these studies offer new insights into the mechanism of allosteric regulation in pyruvate carboxylase and provide an example of how divergent intrinsic properties in homologous enzymes can give rise to their unique regulatory properties.

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