The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties.
The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.
Urbina
J A
JA
Centro de Biología Celular, Facultad de Ciencias, Universidad Central de Venezuela, Caracas.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development
D013439:Sulfhydryl Reagents; D008345:Manganese; D000255:Adenosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)