A biochemical and genetic study of Leishmania donovani pyruvate kinase.
Here we present a biochemical and molecular biology study of the enzyme pyruvate kinase (PYK) from the parasitic protozoa Leishmania donovani. The PYK gene was cloned, mutagenised and over expressed and its kinetic parameters determined. Like in other kinetoplastids, L. donovani PYK is allosterically stimulated by the effector fructose 2,6 biphosphate and not by fructose 1,6 biphosphate. When the putative effector binding site of L. donovani PYK was mutagenised, we obtained two mutants with extreme kinetic behavior: Lys453Leu, which retained a sigmoidal kinetics and was little affected by the effector; and His480Gln, which deployed a hyperbolic kinetics that was not changed by the addition of the effector. Molecular Dynamics (MD) studies revealed that the mutations not only altered the effector binding site of L. donovani PYK but also changed the folding of its domain C.
Sandoval
Will
W
Centro de Biotecnologia, Instituto de Estudios Avanzados, Ministerio de Ciencia y Tecnologia de Venezuela, Carretera Nacional Hoyo de la Puerta, Caracas 1080 A, Venezuela.
Isea
Raúl
R
Rodriguez
Evelyn
E
Ramirez
Jose Luis
JL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D016678:Genome; D006019:Glycolysis; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics*; D007898:Leishmaniasis, Visceral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D006593:Hexokinase; D011770:Pyruvate Kinase