Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism

In metabolism research, thermodynamics is usually used to determine the directionality of a reaction or the feasibility of a pathway. However, the relationship between thermodynamic potentials and fluxes is not limited to questions of directionality: thermodynamics also affects the kinetics of reactions through the flux-force relationship, which states that the logarithm of the ratio between the forward and reverse fluxes is directly proportional to the change in Gibbs energy due to a reaction (Delta(r)G '). Accordingly, if an enzyme catalyzes a reaction with a Delta(r)G ' of -5.7 kJ/mol then the forward flux will be roughly ten times the reverse flux. As Delta(r)G ' approaches equilibrium (Delta(r)G ' = 0 kJ/mol), exponentially more enzyme counterproductively catalyzes the reverse reaction, reducing the net rate at which the reaction proceeds. Thus, the enzyme level required to achieve a given flux increases dramatically near equilibrium. Here, we develop a framework for quantifying the degree to which pathways suffer these thermodynamic limitations on flux. For each pathway, we calculate a single thermodynamically-derived metric (the Max-min Driving Force, MDF), which enables objective ranking of pathways by the degree to which their flux is constrained by low thermodynamic driving force. Our framework accounts for the effect of pH, ionic strength and metabolite concentration ranges and allows us to quantify how alterations to the pathway structure affect the pathway's thermodynamics. Applying this methodology to pathways of central metabolism sheds light on some of their features, including metabolic bypasses (e.g., fermentation pathways bypassing substrate-level phosphorylation), substrate channeling (e.g., of oxaloacetate from malate dehydrogenase to citrate synthase), and use of alternative cofactors (e.g., quinone as an electron acceptor instead of NAD). The methods presented here place another arrow in metabolic engineers' quiver, providing a simple means of evaluating the thermodynamic and kinetic quality of different pathway chemistries that produce the same molecules. Author Summary Given data about enzyme kinetics and reaction thermodynamics, traditional metabolic control analysis (MCA) can pinpoint the enzymes whose expression will have the largest effect on steady-state flux through the pathway. These analyses can aid experimentalists in tuning enzyme expression levels along a metabolic pathway. In this work, we offer a framework that is complementary to MCA. Rather than focusing on the relationship between enzyme levels and pathway flux, we examine a pathway's stoichiometry and thermodynamics and ask whether it is likely to support high flux in cellular conditions. Our framework calculates a single thermodynamically-derived metric (the MDF) for each pathway, which is convenient for selecting the promising pathways from a large collection. This approach has several advantages. First, enzyme kinetic properties are laborious to measure and differ between organisms and isozymes, but no kinetic data is required to calculate the MDF. Second, as our framework accounts for pH, ionic strength and allowed concentration ranges, it is simple to model the effect of these parameters on the MDF. Finally, as it can be difficult to control the exact expression level of enzymes within cells, the MDF helps identify alternative pathways that are less sensitive to the levels of their constituent enzymes.