Abstract
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.
Highlights
Reliable and predictable microbial biocatalysts are a prerequisite for the transition from a petro-based to a green bio-based economy
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
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
Summary
Reliable and predictable microbial biocatalysts are a prerequisite for the transition from a petro-based to a green bio-based economy. It is not surprising that this influence can significantly change both the thermodynamic state variables and kinetic parameters of the reactions [3,6,29] compared to pure buffer solutions Another important factor that has often been neglected is the reversibility of some glycolytic reactions. To investigate the influence of cytosolic conditions, measurements were performed at different temperatures, pH values, concentrations of different salts, and with macromolecular crowding agents. At the highest concentration of crowding agents (250 g kg−1) the reaction enthalpy change decreased to 70 ± 3% (PEG 20,000), to 69 ± 1% (PEG 6000) and to 66 ± 9% (BSA). There is a reduction in the dielectric constant of the solution by the addition of crowding agents, which leads to stronger long-range electrostatic forces between the metabolites This increases the influence of the ionic strength on the activity coefficients and on the reaction enthalpy change. The interaction of these 3 effects could explain the effect of crowding on the reaction enthalpy change
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