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

From the ¹ From the Department of Biochemistry, Western Reserve University, School of Medicine, Cleveland, Ohio 44106

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 (K_exp) 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 K_exp. For the reaction catalyzed by P-enolpyruvate carboxytransphosphorylase (pyrophosphorylase:oxalacetate carboxylase, EC 4.1.1.38), K'_ionic expressed as (oxalacetate^2-) (HPP^3-) / (P-enolpyruvate^3-) (HP_i^2-)(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 (oxalacetate^2-) (ITP^4-)/(P-enolpyruvate^3-)(IDP^3-)(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 (oxalacetate^2-) (ADP^3-)(HP_i^2-)(H⁺)/(pyruvate^1-)(HCO₃^-)(ATP^4-) is (1.4 ± 0.4) x 10^-6. Equilibrium constants for other conditions have been calculated from K_ionic. These constants are expressed in terms of total concentrations of each reactant and are designated K'_anal· K'_anal corresponds to the K_exp 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 F'_anal (F'_anal = -RT In K'_anal) of the linked reaction with ATP and PP_i 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 PP_i. 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 F'_anal is -8.0 ± 0.3 kcal and with Mg²⁺ at 5 mm, the F'_anal is -4.5 ± 0.3 kcal.

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