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

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

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Free PMC article

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

Jorge Hidalgo et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Double reciprocals and secondary plots of 3-MP adding PEP and GDP as variable substrates.
(A-C) Double reciprocal, slopes and intercepts of 139Met varying PEP. (D-F) Double reciprocal, slopes and intercepts of 139Leu varying PEP. (G) Double reciprocal of 139Met varying GDP. (H) Double reciprocal of 139Leu varying GDP. 139Met and 139Leu were assayed as described in Materials and Methods with increasing concentration of substrates (50–600 μM for PEP, 10–100 μM for GDP) and 3-MP concentration between 10–100 μM.
Fig 2
Fig 2. Double reciprocals and secondary plots of 3-MP adding OAA and GTP as variable substrates.
(A-C) Double reciprocal, slopes and intercepts of 139Met varying OAA. (D-F) Double reciprocal, slopes and intercepts of 139Leu varying OAA. (G) Double reciprocal of 139Met varying GTP. (H) Double reciprocal of 139Leu varying GTP.139Met and 139Leu were assayed as described in Materials and Methods with increasing concentration of substrates (5–50 μM for OAA, 25–500 μM for GTP) and 3-MP concentration between 10–100 μM for OAA and between 50–400 μM for GTP.
Fig 3
Fig 3. Thermal unfolding of PEPCK-C in presence and absence of compound 1 or 2.
Fig 4
Fig 4. Structural formula of compounds 1 and 2.
Fig 5
Fig 5. Double reciprocal plots of compound 1 inhibition varying glyceroneogenic substrates.
Fig 6
Fig 6. Double reciprocal plots of compound 2 inhibition varying glyceroneogenic substrates.
Fig 7
Fig 7. Near-UV CD spectra of PEPCK-C isoenzymes.
50 μM 3-MP (A-B), compound 1 (C-D) or compound 2 (E-F) were added to 20 μM PEPCK-C in a 20 mM HEPES pH 7.4 buffer and 1 mM TCEP.
Fig 8
Fig 8. ITC measurements of PEPCK-C isoenzymes with 3-MP or compound 2.
The upper part of each panel shows the thermogram after baseline correction, and the lower part shows the binding isotherm with the fit to a single class of binding sites model. (A) 139Met with 3-MP. (B) 139Met with compound 2.

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Grant support

This study was supported by research grants AGL2008-01487ALI (www.mineco.gob.es), DGA-IAF FITE2012/2013 (www.aragob.es), and UZ2014-CIE-03 (www.unizar.es) to P.L.B., AGL2015-66177 to P.L.B. and J.A.C., and grants BFU2013-47064-P (www.micinn.es), BIO2014-57314-REDT (www.mineco.gob.es) 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.
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