Abstract 1822 Assessment of Allosteric Inhibition of Pyruvate Carboxylase by Phenylacetate and Phenylacetyl-CoA

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, a key TCA cycle intermediate critical in gluconeogenesis, fatty acid synthesis, and amino acid synthesis.Elevated PC activity is correlated with fasting hyperglycemia in type-2 diabetes and the growth and metastasis of cancerous cells.Consequently, research efforts have increasingly focused on identifying potent inhibitors of this allosteric enzyme.PC is subjected to extensive allosteric regulation and is activated by acetyl-CoA in most organisms.However, a prior published study has shown that aromatic analogues of acetyl-CoA are inhibitors, rather than activators, of PC.Consistent with several studies that have shown phenylacetate to reduce gluconeogenic flux in both yeast and vertebrate systems, this earlier study suggested that phenylacetate is intracellularly converted to phenylacetyl-CoA, which directly inhibits rat liver PC.However, the mechanism of inhibition for this aromatic acid and its CoA thioester remains undefined.To follow on these previously published results, the current study investigates the inhibitory potential of aromatic acids and their acyl-CoA thioesters (phenylacetate & phenylacetyl-CoA, phenylpropionate & phenylpropionyl-CoA, and benzoate & benzoyl-CoA) on PC purified from Staphylococcus aureus (SaPC), Aspergillus nidulans (AnPC), and Sus scrofa (SsPC).The initial hypothesis is that these compounds bind to the allosteric site of PC and displace the allosteric activator, acetyl-CoA.Using a malate dehydrogenase coupled enzyme assay, we have determined that these three aromatic acids inhibit PC from all three source organisms.Phenylacetate displays an IC50 value of 7.3 mM, 6.5 mM, and 20.7 mM for SaPC, SsPC, and AnPC, respectively.Consistent with reports that used rat liver PC, the aromatic CoA thioesters inhibit PC purified from pig liver (SsPC).Interestingly, the aromatic CoA thioesters appear to activate, rather than inhibit, bacterial SaPC.The data currently suggests that aromatic CoA thioesters are weak allosteric activators of bacterial PC from S. aureus and have no inhibitory effects on the fungal PC from A. nidulans.Overall, our data indicate that these compounds are likely binding at the allosteric site and that phenylacetate, rather than phenylacetyl-CoA, is most likely inhibiting PC when this compound is employed in cellular inhibition studies.Further experiments will serve to fully elucidate the mechanism of inhibition of these aromatic compounds.