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Keywords:

  • Citrate synthase. C4 metabolism;
  • coupled enzyme assays;
  • crassulacean acid metabolism;
  • malate dehydrogenase;
  • phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable Km values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the Vmax value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, Vmax is brought back to the same level as that with the NADH-coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose-6-phosphate in concentrations higher than 1 mM blocks the response of PEPC to added MDH in the CS assay.