Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites.
We have cloned a cDNA encoding Toxoplasma gondii pyruvate kinase and obtained the full-length recombinant enzyme with a calculated molecular mass of 57.5 kDa. The predicted amino acid sequence of T. gondii pyruvate kinase exhibited a highest identity (63%) to that of Eimeria tenella pyruvate kinase and a lower identity of less than 25% to the pyruvate kinases from other organisms. Southern blot analysis indicated that the pyruvate kinase gene existed as a single copy in the T. gondii tachyzoite. The active recombinant enzyme contained four subunits and produced a strongly sigmoid saturation curve with phosphoenolpyruvate as the variable substrate. Fructose 1,6-diphosphate, a general activating factor of pyruvate kinase in most species, did not affect the enzyme activity. However, glucose 6-phosphate radically activated the enzyme. Fructose 2,6-diphosphate suppressed the reaction velocity at a higher concentration of phosphoenolpyruvate. These properties indicate that pyruvate kinase activity in T. gondii is regulated by unusual phosphorylated sugars.
Maeda
Takuya
T
Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Saito
Tomoya
T
Oguchi
Yoshiyo
Y
Nakazawa
Miki
M
Takeuchi
Tsutomu
T
Asai
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D019298:Glucose-6-Phosphate / Q000378:metabolism; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D010728:Phosphoenolpyruvate / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014122:Toxoplasma / Q000201:enzymology* / Q000254:growth & development* / Q000502:physiology
D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019298:Glucose-6-Phosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase