Inhibition of phosphoenolpyruvate carboxykinase from Trypanosoma (Schizotrypanum) cruzi epimastigotes by 3-mercaptopicolinic acid: in vitro and in vivo studies.
3-mercaptopicolinic acid (3MP) was shown to be a powerful and specific inhibitor of the phosphoenolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxyloacetate carboxylyase (transphosphorylating), EC 4.1.1.49) isolated and purified to homogeneity from Trypanosoma (Schizotrypanum) cruzi epimastigotes (Urbina, J. A., 1987, Arch. Biochem. Biophys. 258, 186-195). In the presence of saturating concentrations of the cosubstrates the inhibition was purely noncompetitive toward all substrates in the carboxylation reaction. The inhibition was specific to this enzyme, being nonexistent or moderate toward eight other enzymes tested that are involved in glycolysis, hexose monophosphate shunt, Krebs' cycle, and amino acid metabolism. These facts, together with the kinetic constants of the enzyme and the intracellular concentrations of its substrates, predicted a very potent inhibition of the reaction catalyzed by this enzyme in vivo. In accordance of this prediction 200 microM 3MP inhibited 2.2-fold the production of [2,2'-13C]succinate from D-[1-13C]glucose by intact epimastigotes under anaerobic conditions, as shown by 13C NMR and 1H NMR spectroscopy; correspondingly the overall glucose consumption rate decreased by the same factor, while the relative rate of production (per mole of glucose consumed) of the other main product of glucose catabolism, [3-13C]alanine, was increased 3-fold by the drug. Under aerobic conditions the glucose catabolism was faster (negative Pasteur effect) and the drug at the same concentration again blocked succinate production but had negligible effects on glucose consumption. On the other hand, 200 microM 3MP blocked completely the epimastigotes' catabolism of L-[U-14C]proline through the Kreb's cycle via PEP-carboxykinase, as indicated by the disappearance of 14C label present in alanine, pyruvate, citrate, and isocitrate after 1 h of incubation in the presence of the labeled amino acid, while the amount of radioactivity present in alpha-ketoglutarate and malate doubled. The results support the proposition that PEP-carboxykinase has a central role in the energy metabolism of this organism as it is essential for the catabolism of amino acids.
Urbina
J A
JA
Centro de Biologia Celular, Escuela de Biologia, Universidad Central de Venezuela, Caracas.
Osorno
C E
CE
Rojas
A
A
D000693:Anaerobiosis; D000818:Animals; D002247:Carbon Isotopes; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008954:Models, Biological; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D010848:Picolinic Acids / Q000494:pharmacology*; D011392:Proline / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000378:metabolism
D002247:Carbon Isotopes; D010848:Picolinic Acids; C008363:3-mercaptopicolinic acid; D011392:Proline; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005947:Glucose