Thermodynamic Parameters for the Hydrolysis of Inorganic Pyrophosphate at pH 7.4 as a Function of [Mg2+], [K+], and Ionic Strength Determined from Equilibrium Studies of the Reaction

• Published in: The Journal of biological chemistry Vol. 249; no. 11; pp. 3465 - 3474
• Main Authors: Flodgaard, H, Fleron, P
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10-06-1974
• Subjects: Animals; Buffers; Chemical Phenomena; Chemistry; Diphosphates - analysis; Diphosphates - metabolism; Evaluation Studies as Topic; Hydrogen-Ion Concentration; Hydrolysis; Liver - analysis; Magnesium; Mathematics; Methods; Osmolar Concentration; Phosphates; Potassium; Rats; Temperature; Thermodynamics; Tritium
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)42596-9

The equilibrium between inorganic pyrophosphate and inorganic orthophosphate was studied by direct measurement of PP i . The reaction was catalyzed by yeast pyrophosphatase in the presence of Mg 2+ ions or by alkaline phosphatase in the absence of metal ions. The concentration of PP i was determined by an isotope derivative method. Identical concentrations of PP i were obtained whether the reaction proceeded from orthophosphate or pyrophosphate provided other conditions were identical.
The equilibrium concentrations were measured at varying Mg 2+ and K + ion concentrations and at different ionic strengths. All measurements were performed at pH 7.4. Equilibrium was established
at different temperatures and the enthalpy changes were determined.
HPP 3- ⇆ 2HP 2- + H +
The experimental data were used to compute an equilibrium constant ( K ion ic ) based on the ionic species
K ion ic was found to (2.41 ± 0.20) x 10 -4 m 2 (zero ionic strength) corresponding to a Δ G ion ic = (4.98 ± 0.05) Cal x mole -1 (20.84 kJ x mole -1 ). It was constant over the variety of conditions studied. From the experiments carried out in absence of metal ions and low
ionic strength Δ G 0' obs was found to (-5.63 ± 0.02) Cal x mole -1 (-23.56kJ x mole -1 ) calculated for zero ionic strength making due allowances for dissociation at pH 7.4. Δ H 0' obs was found to (-1.90 ± 0.37) Cal x mole -1 (-7.95 kJ x mole -1 ) and T Δ S 0' obs to 3.87 Cal x mole -1 (16.19 kJ x mole -1 ) (the latter two results given for ionic strength 0.127). Increasing the Mg 2+ ion concentration makes the reaction more unfavorable, thus at 26 m m free [Mg 2+ ] and 18 m m free [K + ] at ionic strength 0.1 Δ G 0' obs was found to (-2.73 ± 0.07) Cal x mole -1 (-11.42 kJ x mole -1 ).
The stoichiometry of the participation of H + and metal ions in the composite reaction was calculated. The contribution of the observed enthalpy and entropy terms to the
variation in Δ G 0' obs is discussed in relation to the changes in production or consumption of H + and metal ions. At free [Mg 2+ ] above 0.3 m m and low free [K + ] (17 to 43 m m ) at ionic strengths around 0.05 the reaction was found to consume acid. The T Δ S 0' obs term which is negative was found to pass through a maximum negative value of -4.17 Cal x mole -1 (-17.53 kJ x mole -1 ) at 12.2 m m free [Mg 2+ ]. The entropy term is numerically considerably larger than the enthalpy term, resulting in a less favorable reaction. Δ H 0' obs values were calculated on the basis of Δ H 0 for dissociation and association reactions and agreement with the experimental results was obtained in the cases where Δ H 0 values reported in the literature were measured at experimental conditions similar to ours.
From Δ G 0' obs measured at the ionic strength and [K + ] and [Mg 2+ ] prevailing in the cell, Δ G for the PP i hydrolysis in the cell was calculated to -4.0 Cal x mole -1 (-16.74 kJ x mole -1 ) on the basis of 1 m m free [Mg 2+ ], 150 m m free [K + ], 2.42 m m free [P i ], and concentrations of PP i measured in freeze biopsies from rat liver to (6.2 ± 0.3 (S.E. n = 6)) nmoles per g wet weight. The implication of this result for the utilization of the free energy of PP i hydrolysis in the cell is discussed.
In an appendix a set of equations were introduced, which permit computation of equilibrium data also in a general case.