Purification and properties of phosphoenolpyruvate carboxylase from Molinema dessetae (Nematoda: Filarioidea).
The presence of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), an enzyme at the branchpoint of glycolysis and the Krebs cycle was detected in the Filaria Molinema dessetae. This enzyme has not previously been identified in Helminths, which have so far been found to only possess a phosphoenolpyruvate carboxykinase (EC 4.1.1.32). This enzyme had a level of activity comparable to that of pyruvate kinase, and was relatively less active than enzymes such as malate dehydrogenase or lactate dehydrogenase. We propose here a method of purification of M. dessetae PEP-carboxylase. When purified to electrophoretic homogeneity, the enzyme had a molecular weight of 64 kDa. Kinetic studies indicated that the carboxylation reaction had an optimal pH of 5.8. The enzyme was inhibited by cations such as Fe2+, Zn2+, Cd2+, Cu2+ but required the presence of Mg2+ or Mn2+. The enzyme was thermostable. The apparent Km value of 2.38 mmol for phosphoenolpyruvate for the carboxylation reaction was higher than previously reported values. The Km value for KHCO3 was found to be 1.6 mmol. PEP-carboxylase did not catalyse the reverse reaction.
Loiseau
P M
PM
Biologie et Contrôle des Organismes Parasites, Université de Paris-Sud, Châtenay-Malabry.
Gayral
P
P
Petek
F
F
D000818:Animals; D002412:Cations / Q000494:pharmacology; D005370:Filarioidea / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007290:Inosine Diphosphate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010071:Oxaloacetates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation
D002412:Cations; D010071:Oxaloacetates; D006153:Guanosine Diphosphate; D010728:Phosphoenolpyruvate; D007290:Inosine Diphosphate; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium