PURIFICATION AND CHARACTERIZATION OF PHOSPHO ENOLPYRUVATE CARBOXYKINASE FROM TRYPANOSOMA-BRUCEI

ATP-dependent phospho enolpyruvate carboxykinase (EC 4.1.1.49; PEPCK, ATP) was purified from glycosomes of cultured procyclic Trypanosoma br ucei to electrophoretic homogeneity. The purified enzyme exhibited a m ean specific activity of 83 units mg(-1) as measured in the carboxylat ion direction at 30 degrees C. A similar activity was obtained for the decarboxylation reaction. The enzyme was shown to be a homodimer in s olution with a subunit molecular mass of 59 kDa. Amino acid sequence a nalysis suggested that the PEPCK (ATP) is identical to the trypanosoma l protein p60, the sequence of which was previously predicted from the corresponding nucleotide sequence by other investigators. The basic n ature of the enzyme was indicated by a high isoelectric point (pH 8.9) . The enzyme was found to be strictly dependent on adenosine nucleotid es for activity, as well as on the presence of Mn2+. Mg2+ was found to be ineffective as activator of the trypanosomal enzyme, but a combina tion of subsaturating (less than or equal to 300 mu M) concentrations of Mn2+ and high concentrations of Mg2+ caused a synergistic effect on the carboxylation activity, indicating a dual cation requirement. Mn2 + is necessary to activate the enzyme and Mn2+ or Mg2+ most likely for ms the cation-nucleotide complex as the active form of the substrate. Relatively high (5 mM) levels of ATP were required to produce a signif icant inhibition of the carboxylation reaction. Quinolinic acid, a str uctural analogue of oxaloacetate, completely inhibited the decarboxyla tion reaction at a 1 mM concentration. The apparent Michaelis constant s of the enzyme were 490 mu M for PEP, 37 mu M for oxaloacetate, 40 mu M for ADP, 10.3 mu M for ATP, 970 mu M for Mn2+ and 26 mM for HCO3-. Endogenous substrate concentrations were found to be 327 nmol PEP, 148 6 nmol ADP, 4200 nmol ATP and 11.5 nmol Mn2+ (ml cell volume)(-1). Our kinetic data suggest that under physiological conditions PEPCK (ATP) in T. brucei is bidirectional and that its activity is regulated prima rily by mass action. The physiological relevance of the enzyme in proc yclic T. brucei is discussed.