Phosphoenolpyruvate carboxykinase from Trypanosoma cruzi. Purification and physicochemical and kinetic properties

Mol Biochem Parasitol. 1995 Jul;73(1-2):91-101. doi: 10.1016/0166-6851(95)00099-m.

Abstract

Phospho enolpyruvate carboxykinase (PEPCK) has been purified to homogeneity from epimastigotes of the Tul 0 strain of Trypanosoma cruzi. The physicochemical parameters determined allowed the calculation of an average molecular mass of 120 kDa; the subunit molecular mass, about 61 kDa, is in good agreement with the value of 58.6 kDa recently determined from the sequence by Sommer et al. (FEBS Lett. 359 (1994) 125-129). The PEPCK from T. cruzi presented, in addition to its molecular mass, typical properties of other ATP-linked PEPCKs, namely strict specificity for ADP in the carboxylation reaction and lower specificity in the decarboxylation and exchange reactions, and synergistic activation by CdCl2 or MgCl2 when added in addition to MnCl2. The enzyme presented hysteretic behaviour, shown by a lag period in the carboxylation reaction, which was affected by dilution and preincubation. The decarboxylation reaction catalyzed by the T. cruzi PEPCK was not inhibited by excess of ATP-Mn. The apparent Km values for the carboxylation reaction, including the low value for PEP (0.035 mM) are compatible with an important role of PEPCK, as suggested by previous NMR experiments, on the CO2 fixation in vivo which leads to succinate excretion during aerobic fermentation of glucose.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cations, Divalent / pharmacology
  • Chemical Phenomena
  • Chemistry, Physical
  • Enzyme Inhibitors / pharmacology
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • NAD / pharmacology
  • Nucleotides
  • Phosphoenolpyruvate Carboxykinase (GTP) / chemistry
  • Phosphoenolpyruvate Carboxykinase (GTP) / isolation & purification*
  • Phosphoenolpyruvate Carboxykinase (GTP) / metabolism
  • Protein Conformation
  • Substrate Specificity
  • Trypanosoma cruzi / enzymology*

Substances

  • Cations, Divalent
  • Enzyme Inhibitors
  • Nucleotides
  • NAD
  • Phosphoenolpyruvate Carboxykinase (GTP)