Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism.
Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.
Callens
M
M
International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases, Brussels, Belgium.
Kuntz
D A
DA
Opperdoes
F R
FR
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D000838:Anions / Q000494:pharmacology; D050260:Carbohydrate Metabolism; D002412:Cations / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects / Q000469:parasitology; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development
D000596:Amino Acids; D000838:Anions; D002412:Cations; D010710:Phosphates; D011770:Pyruvate Kinase