Purification and properties of pyruvate kinase from Streptococcus lactis.
The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) of Streptococcus lactis C10 is activated by fructose 1,6-diphosphate (Fru-1,6-P2), activity being a sigmoidal function of activator concentration. The FDP0.5V (Fru-1,6-P2 concentration giving half-maximal velocity) is markedly increased in the presence of low concentrations of inorganic phosphate; 1 mM phosphate increases the FDP0.5V value 6-fold. Although the intracellular level of Fru-1,6-P2 (12-18 mM) in exponentially growing cells on the medium used is much greater than the FDP0.5V for pyruvate kinase (0.2 mM) as determined in triethanolamine-HCl buffer, a much higher Fru-1,6-P2 concentration may be required to activate the enzyme in vivo to overcome phosphate inhibition. Tris and maleate also inhibit the enzyme. At low concentrations of Fru-1,6-P2 (0.1 mM), reaction rate is a sigmoidal function of both phosphoenolpyruvate and adenosine diphosphate (ADP) concentrations; at near saturating concentrations of activator (1 mM) the response to varying ADP is hyperbolic while the response to varying phosphoenolpyruvate becomes much less sigmoidal. The affinity for both substrates (especially phosphoenolpyruvate) is also increased by increasing the concentration of Fru-1,6-P2. The affinity of the enzyme for guanosine disphosphate (GDP) is 12-13 times that for ADP under the assay conditions used. The Streptococcus lactis pyruvate kinase has a molecular weight of 240000 with a subunit molecular weight of 60000.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology; D008298:Maleates / Q000494:pharmacology; D008970:Molecular Weight; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*
D005636:Fructosephosphates; D006150:Guanine Nucleotides; D008298:Maleates; D010710:Phosphates; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase