STRUCTURAL AND FUNCTIONAL ANALYSIS OF AROMATIC COMPOUNDS AS ALLOSTERIC INHIBITORS OF PYRUVATE CARBOXYLASE
Date of Award
Summer 7-21-2026
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biological Sciences
First Advisor
Martin St. Maurice
Second Advisor
Anita Manogaran
Third Advisor
Emily Sontag
Fourth Advisor
Martin St. Maurice
Abstract
Pyruvate carboxylase (PC) is an anaplerotic enzyme that replenishes TCA cycle intermediates by converting pyruvate to oxaloacetate. PC plays essential roles in central metabolism and is allosterically activated by acetyl-CoA and allosterically inhibited by dicarboxylic acids such as L-aspartate and L-glutamate. Dysregulation and overexpression of PC have been linked with type-2 diabetes, cancer progression, and microbial infections, making the enzyme a therapeutic target. An aromatic metabolite, phenylacetate, was widely used as a presumed PC inhibitor in cellular studies. It was thought to convert to the CoA thioester form, which inhibited PC either by competing with or by depleting cellular pools of allosteric activator acetyl-CoA. However, the exact binding site of phenylacetate on PC or mechanism of inhibition has not been rigorously characterized. This thesis aimed to characterize the mechanistic and structural features of phenylacetate as a PC inhibitor. Contrary to the prevailing view that phenylacetate acts via conversion to phenylacetyl-CoA, this study demonstrates that phenylacetate directly inhibits purified PC across multiple domains of life. Kinetic analyses indicate that phenylacetate shares a binding site with acetyl-CoA and L-aspartate on bacterial PC, suggesting a binding location at the allosteric site, where acetyl-CoA and L-aspartate binding have been previously reported. Our study also shows that phenylacetyl-CoA inhibits vertebrate PC but activates bacterial PC, suggesting a species-specific inhibition mechanism for acyl-CoA thioesters. This study further extends the search for a suitable allosteric inhibitor of PC by implementing the structure-activity relationship (SAR) of analogues derived from phenylacetate and dicarboxylic amino acids. These analyses identified key structural features required for inhibition and revealed several polar analogues with enhanced inhibitory potency and membrane permeability characteristics. Finally, to probe the binding location of phenylacetate at the allosteric site, mutagenesis and kinetic studies identified Arg21 and Arg414 as important contributors to phenylacetate binding, providing structural insight into the binding mechanism. Taken together, this study establishes phenylacetate as a direct allosteric inhibitor of PC and provides new insights into the molecular basis of allosteric regulation at the BC dimer interface. The results also provide a foundation for the rational drug design of selective inhibitors.