Inhibition and Allosteric Regulation of Monomeric Phosphoenolpyruvate Carboxykinase by 3-Mercaptopicolinic Acid

Abstract

For almost 40 years, it has been known that tryptophan metabolites and picolinic acid analogues act as inhibitors of gluconeogenesis. Early studies observed that 3-mercaptopicolinic acid (MPA) was a potent hypoglycemic agent via inhibition of glucose synthesis through the specific inhibition of phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenesis pathway. Despite prior kinetic investigation, the mechanism of the inhibition by MPA is unclear. To clarify the mechanism of inhibition exerted by MPA on PEPCK, we have undertaken structural and kinetic studies. The kinetic data in concert with crystallographic structures of PEPCK in complex with MPA and the substrates for the reaction illustrate that PEPCK is inhibited by the binding of MPA at two discrete binding sites: one acting in a competitive fashion with PEP/OAA (∼10 μM) and the other acting at a previously unidentified allosteric site (Ki ∼ 150 μM). The structural studies suggest that binding of MPA to the allosteric pocket stabilizes an altered conformation of the nucleotide-binding site that in turn reduces the affinity of the enzyme for the nucleotide.

Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors

“…Whereas n was 1 in all experiments using compound 2, we found a value of 2 when using 3-MP. As this parameter reflects the number of binding sites, our results suggest that there is only one binding site for compound 2 but two sites for 3-MP, in agreement with two 3-MP binding sites reported in the crystallographic structure of rat PEPCK-C by Balan et al [ 23 ].…”

“…The existence of notable differences between K d values for both isoenzymes in the presence and in the absence of GTP ( Table 4 ) confirms that both inhibitors affect the nucleotide binding site. The sites of 3-MP binding have been already determined by Balan et al [ 23 ]; our results with compound 2 do not allow us to specify where the binding site for this inhibitor is located.…”

“…This inhibition pattern requires a prior binding of the substrate to the active site in order to make the inhibitor site accessible. Recently, Balan et al [ 23 ] found a very similar result studying the inhibition of rat PEPCK-C by 3-MP using both kinetic experiments and the determination of the 3D structure of the protein with bound 3-MP. They find uncompetitive inhibition when using GDP or GTP as variable substrates.…”

“…These structural data are not compatible with the apparent uncompetitive character of 3-MP inhibition because uncompetitive inhibition requires the existence of independent (or different) substrate and inhibitor binding sites [ 24 ]. Balan et al [ 23 ] explain their results suggesting that the anomalous uncompetitive inhibition detected by kinetic means is due to the communication between the allosteric inhibitor site and the active site. Whatever is the true nature of the inhibition by 3-MP, this inhibitor serves to demonstrate the existence of further biochemical differences between pig PEPCK-C isoenzymes because the comparison of K i values for 3-MP in the presence of GTP showed strong differences between pig isoenzymes.…”

c.A2456C-substitution in Pck1 changes the enzyme kinetic and functional properties modifying fat distribution in pigs

“…Despite the substitution being located far away from the active center of the protein, in a region of about 20 amino acids highly conserved from C. elegans to man, our results indicate that it is responsible for catalytic differences in this enzyme. These long-range effects are at the basis of allosteric phenomena in proteins, which had not been described in Pck1 until the very recent report by Balan and coworkers 27 .…”