The Equilibria of Reactions Catalyzed by Carboxytransphosphorylase, Carboxykinase, and Pyruvate Carboxylase and the Synthesis of Phosphoenolpyruvate

The possible role of inorganic pyrophosphate as an energy source for synthesis of phosphoenolpyruvate was evaluated by determining the equilibrium constants of three reactions in which CO2 is fixed into oxalacetate. The experimental constants (Kexp) were determined on the basis of total concentrations of reactants. Then K'ionic for 25°, 0.1 ionic strength, and selected ionic species was estimated from Kexp. For the reaction catalyzed by P-enolpyruvate carboxytransphosphorylase (pyrophosphorylase:oxalacetate carboxylase, EC 4.1.1.38), K'ionic expressed as (oxalacetate2-) (HPP3-) / (P-enolpyruvate3-) (HPi2-)(HCO3-) (H+) was found to be (9 ± 2) x 107. For the reaction catalyzed by P-enolpyruvate carboxykinase (guanosine triphosphate:oxalacetate carboxylase, EC 4.1.1.32), K'ionic expressed as (oxalacetate2-) (ITP4-)/(P-enolpyruvate3-)(IDP3-)(HCO3-) (H+) is (2.2 ± 0.5) x 107. With pyruvate carboxylase (pyruvate:CO2 ligase (adenosine diphosphate), EC 6.4.1.1), K'ionic expressed as (oxalacetate2-) (ADP3-)(HPi2-)(H+)/(pyruvate1-)(HCO3-)(ATP4-) is (1.4 ± 0.4) x 10-6. Equilibrium constants for other conditions have been calculated from Kionic. These constants are expressed in terms of total concentrations of each reactant and are designated K'anal· K'anal corresponds to the Kexp which would be observed under the selected conditions. It is shown that variations of the Mg2+ concentration and pH affect each equilibrium differently. Pyruvate carboxylase and P-enolpyruvate carboxykinase, when linked, catalyze the formation of P-enolpyruvate from pyruvate with utilization of ATP and ITP. Likewise, it has been observed that pyruvate carboxylase and carboxytransphosphorylase may be linked and that P-enolpyruvate is synthesized from pyruvate with utilization of ATP and inorganic pyrophosphate. The ΔF'anal (ΔF'anal = -RT In K'anal) of the linked reaction with ATP and PPi is not as favorable for synthesis of P-enolpyruvate as it is with ATP and ITP. At 0.5 mm Mg2+, 0.1 ionic strength, and pH 8.0, it is 0.2 ± 0.3 kcal compared to -2.3 ± 0.3 kcal for the linked reaction with ATP and ITP. A low concentration of Mg2+ and an alkaline pH is most favorable for the synthesis with PPi. The linked reactions with carboxytransphosphorylase may provide a mechanism for the utilization of the energy of inorganic pyrophosphate. The free energy of hydrolysis of inorganic pyrophosphate has been estimated as a function of Mg2+ concentration and pH. At 0.1 ionic strength, 25°, and pH 7.0, and in the absence of Mg2+, the ΔF'anal is -8.0 ± 0.3 kcal and with Mg2+ at 5 mm, the ΔF'anal is -4.5 ± 0.3 kcal.