How reliable are thermodynamic feasibility statements of biochemical pathways?

Abstract

The driving force for organo- or lithotrophic growth as well as for each step in the metabolic network is the Gibbs reaction energy. For each enzymatic step it must be negative. Thermodynamics contributes therefore to the in-silico description of living systems. It may be used for assessing the feasibility of a given pathway because it provides a further constraint for those pathways which are feasible from the point of view of mass balance calculations (metabolic flux analysis) and the genetic potential of an organism. However, when this constraint was applied to lactic acid fermentation according to a method proposed by Mavrovouniotis (1993a, ISMB 93:273-283) it turned out that an unrealistically wide metabolite concentration range had to be assumed to make this well-known glycolytic pathway thermodynamically feasible. During a search for the reasons of this surprising result the insufficient consideration of the activity coefficients was identified as main cause. However, it is shown in the present contribution that the influence of the activity coefficients on Gibbs reaction energy can be easily taken into account based on the intracellular ionic strength. The uncertainty of the tabulated equilibrium constants and of the apparent standard Gibbs energies derived from them was found to be the second most important reason for the erroneous result of the feasibility analysis. Deviations of intracellular pH from the standard value and bad estimations of currency metabolites, e.g., NAD(+) and NADH, were found to be of lesser importance but not negligible. The pH dependency of Gibbs reaction enthalpy was proved to be easily taken into account. Therefore, the application of thermodynamics for a better in-silico prediction of the behavior of living cell factories calls predominantly for better equilibrium data determined under well defined conditions and also for a more detailed knowledge about the intracellular ionic strength and pH value.

Influence of uncertainties in pH, pMg, activity coefficients, metabolite concentrations, and other factors on the analysis of the thermodynamic feasibility of metabolic pathways

“…Values of the standard Gibbs energies of reaction are either calculated from the experimentally measured equilibrium constants, or estimated using group contributions methods (Forsythe et al, 1997;Henry et al, 2006Henry et al, , 2007Maskow and von Stockar, 2005;Mavrovouniotis, 1990Mavrovouniotis, , 1993bPissarra and Nielsen, 1997).…”

“…A and B are universally valid constants with values of 0.5093 and 1.6 mol À1/2 L 1/2 , respectively (Maskow and von Stockar, 2005). Substituting Equation (8) into Equation (7c) yields Equation (9), which relates the apparent standard reaction Gibbs energy to the standard reaction Gibbs energy at standard state (i.e., infinite dilution)…”

“…Newly available data and the awareness of the importance of the contributions that thermodynamics can provide resulted in several scientific papers dealing with what is becoming known as biochemical thermodynamics. These advances include: (a) biomass yield prediction (Liu et al, 2007;von Stockar et al, 2008), (b) maintenance coefficient prediction (Tijhuis et al, 1993), (c) maximum growth rates and threshold concentration estimation (Heijnen, 1999), (d) bioprocesses kinetics using non-equilibrium thermodynamics, and (e) analysis of metabolic networks (Beard et al, 2002;Henry et al, 2007;Maskow and von Stockar, 2005;Pissarra and Nielsen, 1997).…”

“…On the other hand, Maskow and von Stockar (2005) pointed out that the results of TFA may seriously be biased by any of the following: neglecting the non-ideality of the reacting medium or correcting it based on the extended Debye-Hückel but using inaccurate ionic strength values, assuming a wrong range of intercellular concentrations, erroneous pH, or using uncertain standard Gibbs energies of reaction. Therefore, the present article investigates in how far such simplifying assumptions have an impact on the conclusions obtained from TFA by analyzing these factors quantitatively.…”

Options for biochemical production of 4‐hydroxybutyrate and its lactone as a substitute for petrochemical production

“…Therefore, it is common practice to use the experimentally determined apparent standard Gibbs free energy (D r G 00 ) instead of D r G 0 (Maskow and von Stockar, 2005). D r G 00 is defined by…”