In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

The design, construction, and calibration of a precision thermometric titration calorimeter is described. This calorimeter was tested by determining the enthalpy change for ionization of water. The result, 13.34 ±0.03 kcal/mole (standard deviation), is in exact agreement with the best literature values. This standard deviation indicates that, using this calorimeter, ΔH values can be determined with an accuracy of ±0.0.5 kcal/mole. A calorimetric procedure has been developed for the, simultaneous determination of the equilibrium constant, the enthalpy change, and the entropy change for a chemical reaction from a single titration (Entropy Titration). This procedure has been tested by determining pK, ΔH, and ΔS for proton ionization from HPO42- and HSO4-. The method has also been used to determine pK, ΔH, and ΔS values for proton ionization from Adenosine and Ribese. The resultant pK tor each system has an accuracy of about ±0.05 pK unit. Enthalpy and entropy changes for proton ionization have been determined for twenty-six carboxylic acids. The results are discussed in terms of three previous approaches (i.e. inductive, electrostatic, and the linear relation between ΔH° and ΔS°). The results are shown not to fit any of these theories well and reasons for this are proposed. An alternate explanation is proposed based on the observed fit of the carboxylic acid data to a linear ΔG° vs ΔS° plot. The site of proton ionization from adenosine is established to be the ribose moiety. It is also shown that both the 2' and 3' hydroxyl groups are necessary for this acidity to exist in aqueous solution.

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