Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum.

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K(m) of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1mM pyridoxal-5'-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K(i) of 0.8mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.