Investigation into pyruvate kinases from Escherichia coli K-12 grown under aerobic and anaerobic conditions.
Two forms of anaerobic Escherichia coli K-12 pyruvate kinase (EC 2.7.1.40) were separated by ammonium sulphate fractionations. Pyruvate kinases I is allosteric and pyruvate kinase II is non-allosteric to phosphoenolpyruvate. The addition of 1 mM FDP reversed the allostery to normal Michaelis-Menten kinetics. AMP had no effect, whereas 8 mM ATP completely inhibited the enzyme. The enzyme showed normal kinetics with ADP as substrate. Mg2+ and Mn2+ stimulated whereas Cu2+ severely inhibited the enzyme, which could be reversed by the addition of 1 mM FDP. Citrate, alpha-ketoglutarate, succinate, fumarate and alanine inhibited the enzyme, whereas phenylalanine had no effect. The allosteric pyruvate kinase from aerobic cultures was not only activated by FDP, but also by AMP. FDP changed Km and Vmax, whereas AMP influenced only the Km. During aerobic-anaerobic transition, pyruvate kinase synthesis increases and reaches a maximum under anaerobic conditions. The degree of FDP activation remains constant, but AMP activation is lost during transition. Aerobic cultures of E. coli K-12 grown on gluconeogenic substrates exhibited pyruvate kinase II activity (non-allosteric), which was stimulated by FDP and by AMP. It has been suggested that E. coli may have two types of pyruvate kinase II depending on the substrate and two types of pyruvate kinase I depending on oxygen tension in the medium.
D000227:Adenine Nucleotides / Q000378:metabolism; D000332:Aerobiosis; D000693:Anaerobiosis; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism*
D000227:Adenine Nucleotides; D005636:Fructosephosphates; D007527:Isoenzymes; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D005947:Glucose