Characterization and properties of the pyruvate phosphorylation system of Acetobacter xylinum.
The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg(2+) ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P(i)) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent K(m) values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; P(i), 0.8 mm; Mg(2+), 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 mum; PP, 0.067 mm; Mg(2+), 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (K(i) = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002262:Carboxy-Lyases / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D005459:Fluorides / Q000037:antagonists & inhibitors; D041822:Gluconacetobacter xylinus / Q000201:enzymology* / Q000378:metabolism; D005943:Gluconeogenesis; D005978:Glutathione / Q000037:antagonists & inhibitors; D006836:Hydro-Lyases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D011770:Pyruvate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism*; D014166:Transferases / Q000378:metabolism
D011773:Pyruvates; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D014166:Transferases; D011770:Pyruvate Kinase; D006867:Hydrolases; D002262:Carboxy-Lyases; D006836:Hydro-Lyases; D005978:Glutathione; D005459:Fluorides