Pyruvate Kinase of the Spore-forming Bacterium, Bacillus Zicheniformis

The kinetic properties of the purified pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from the spore-forming bacterium Bacillus licheniformis were investigated. Catalysis by this enzyme in vitro required both monovalent and divalent cations and proceeded optimally at pH 7.0 to 7.4. The activity of purified pyruvate kinase was modulated by substrate activation (both P-enolpyruvate and ADP), activation by AMP, and inhibition by ATP, Pi, and carbamyl phosphate. Positive cooperativity was shown for P-enolpyruvate, ADP, ATP, and Pi. The inhibition by ATP yielded sigmoidal saturation curves for both P-enolpyruvate and ADP in the presence of Mg(I1) ions. AMP relieved the inhibition caused by ATP, yielding hyperbolic saturation curves for both substrates. The apparent substrate K,,, values, 0.2 mM for P-enolpyruvate, and 0.7 mu for ADP, for the fully activated enzyme in the presence of Mg(I1) were not dependent on the concentration of the second substrate. The kinetic properties observed with Mn(I1) as the activating divalent cation were somewhat different than those observed in the presence of Mg(I1). The apparent K, for P-enolpyruvate was 7-fold lower in the presence of Mn(I1) than the value obtained in the presence of Mg(I1). Furthermore, the inhibition of the Mn(II)activated enzyme by ATP did not alter the hyperbolic nature of the P-enolpyruvate saturation curve and was not relieved by AMP. Therefore, the K type allosteric properties that were observed with Mg(I1) as the activating divalent cation were not manifested in the presence of Mn(II).