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

PLoS One. 2016 Jul 8;11(7):e0159002. doi: 10.1371/journal.pone.0159002. eCollection 2016.


There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

MeSH terms

  • Animals
  • Enzyme Inhibitors / chemistry*
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / chemistry
  • Phosphoenolpyruvate Carboxykinase (GTP) / antagonists & inhibitors*
  • Phosphoenolpyruvate Carboxykinase (GTP) / chemistry*
  • Picolinic Acids / chemistry*
  • Recombinant Proteins / chemistry
  • Swine


  • Enzyme Inhibitors
  • Isoenzymes
  • Picolinic Acids
  • Recombinant Proteins
  • 3-mercaptopicolinic acid
  • Phosphoenolpyruvate Carboxykinase (GTP)

Grant support

This study was supported by research grants AGL2008-01487ALI (, DGA-IAF FITE2012/2013 (, and UZ2014-CIE-03 ( to P.L.B., AGL2015-66177 to P.L.B. and J.A.C., and grants BFU2013-47064-P (, BIO2014-57314-REDT ( and PI078/08 to J.S. P.L. was supported by a predoctoral fellowship of Fundación La Caixa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.