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The glycolytic pathway is one of the most studied metabolic pathways to date. This work focuses on determining the standard Gibbs energy of reaction (Δ R  g  0 ) of the first adenosine triphosphate-yielding reaction step of glycolysis, namely, the 3-phosphoglycerate kinase (PGK) reaction. Trustworthy values of Δ R  g  0 are required for thermodynamic approaches to determine single reaction conversions or even fluxes of metabolic reactions. In literature, the observed Δ R  g  0,obs values are usually determined directly from the experimental equilibrium composition data without accounting for the nonideality of the reaction mixture. That is the reason why the observed Δ R  g  0,obs values do not present consistent standard data as they are a function of the concentration, pH, and pMg. In this work, a combination of experimentally determined equilibrium composition data and activity coefficients of the reacting agents was used to determine Δ R  g  0 values for the temperatures 303, 313, and 323 K at pH 7. The activity coefficients were predicted with the thermodynamic model electrolyte perturbed-chain statistical associating fluid theory (ePC-SAFT). The ePC-SAFT parameters were taken from literature or fitted to new experimental osmotic coefficients. At 313.15 K, a value for Δ R  g  0 of -16.2 ± 0.2 kJ/mol was obtained. This value is about 4 kJ/mol less negative than what is usually considered as an accepted standard value. The reason behind this discrepancy was found to be the activity coefficients of the reacting agents, which dramatically influence the equilibrium position of the PGK reaction. On the basis of the temperature-dependent Δ R  g  0 values, the standard enthalpy of reaction was determined and found to be Δ R  h  0 = -49 ± 9 kJ/mol.

Standard Gibbs Energy of Metabolic Reactions: III The 3-Phosphoglycerate Kinase Reaction