Purification and characterization of a metabolite-regulated pyruvate kinase from Leishmania major promastigotes.

The pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) of Leishmania major promastigotes is a multimer of 59 kDa subunits having an Mr 181000. It is activated by its substrate phosphoenolpyruvate (PEP) in a positively cooperative manner, and heterotropically by fructose 1,6-bisphosphate (FBP). Kinetics with regard to the phosphate acceptor adenosine 5'-diphosphate (ADP), MgCl2, and KCl are hyperbolic and unaffected by FBP. The enzyme is strongly inhibited by the reaction product ATP, as well as GTP and ITP, and to a lesser degree by citrate. Of seven amino acids reported to inhibit the pyruvate kinases of other organisms, none have any effect on the L. major pyruvate kinase in vitro. The enzyme shows its maximum activity at pH 7.0 in the absence of FBP, and at pH 7.6 in its presence. Contrary to previous suggestions, the enzyme appears to be well-suited for a regulatory role in the metabolism of an aerobic organism capable of net glucose synthesis.