Regulation of the pyruvate kinase from Alcaligenes eutrophus H 16 in vitro and in vivo.
The biosynthesis of the enzyme pyruvate kinase (E.C. 2.7.1.40) of Alcaligenes eutrophus (Hydrogenomonas eutropha) H 16 was influenced by the carbon and energy source. After growth on gluconate the specific enzyme activity was high while acetate grown cells exhibited lower activities (340 and 55 mumoles/min-g protein, respectively). The pyruvate kinase from autotrophically grown cells was purified 110-fold. The enzyme was characterized by homotropic cooperative interactions with the substrate phosphoenolpyruvate, the activators AMP, ribose 5-phosphate, glucose-6-phosphate and the inhibitor ortho-phosphate. In addition to phosphate ATP caused inhibition but in this case nonsigmoidal kinetics was obtained. The half maximal substrate saturation constant S0.5 for phosphoenolpyruvate in the absence of any effectors was 0.12 mM, in the presence of 1 mM ribose-5-phosphate 0.07 mM, and with 9 mM phosphate 0.67 mM. The corresponding Hill values were 0.96, 1.1 and 2.75. The ADP saturation curve was hyperbolic even in the presence of the effectors, the Km value was 0.14 mM ADP. When the known intracellular metabolite concentrations in A. eutrophus H 16 were compared with the regulatory sensitivity of the enzyme, it appeared that under the conditions in vivo the inhibition by ATP was more important than the regulation by the allosteric effectors.
D000085:Acetates / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000421:Alcaligenes / Q000201:enzymology* / Q000254:growth & development; D000494:Allosteric Regulation; D004789:Enzyme Activation; D005942:Gluconates / Q000378:metabolism; D005958:Glucosephosphates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology
D000085:Acetates; D005942:Gluconates; D005958:Glucosephosphates; D010710:Phosphates; D012267:Ribosemonophosphates; D000249:Adenosine Monophosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium