Living evidence reveals how thermodynamics shape enzyme investment in glycolysis

protein costs of glycolysis across Z. mobilis, E. coli, and C. thermocellum. Panel A displays bar charts with overlaid line graphs showing protein costs and cumulative delta G for individual enzymes in each species' glycolytic pathway. Panel B compares total protein costs between core glycolysis (G6P to pyruvate) and glycolysis plus periplasmic sugar uptake, with C. thermocellum showing the highest costs (~2400-3200 μg protein/mmol glucose·h⁻¹), followed by E. coli (~1050-1280), and Z. mobilis (~600-650)
In vivo protein costs for glycolytic reactions and pathways. (A) Protein costs, expressed as the amount of protein (μg) required per unit flux (mmol h−1), for glycolytic reactions in Z. mobilis (blue), E. coli (pink), and C. thermocellum (green). (B) The total protein cost, expressed as the amount of protein (μg) required per unit flux (mmol glucose h−1), for core glycolysis (G6P to PYR) and glycolysis including periplasmic sugar uptake in each bacterium.
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Background/Objective

Computational studies predict that thermodynamically constrained reactions and pathways impose greater protein demands on cells, requiring a larger amount of enzyme to sustain a given flux compared to those with stronger thermodynamics. This study provides in vivo evidence that thermodynamic driving forces are a key parameter influencing the enzyme burden of metabolic pathways.

Approach

Scientists with the Great Lakes Bioenergy Research Center and the Center for Bioenergy Innovation quantified absolute concentrations of glycolytic enzymes in three bacterial species (Zymomonas mobils, Escherichia coli, and Clostridium thermocellum) that employ distinct glycolytic pathways with varying thermodynamic driving forces and integrated enzyme data with corresponding in vivo metabolic fluxes and ∆G measurements.

Results

Results showed the more thermodynamically favorable Entner-Doudoroff pathway in Z. mobilis requires one fourth as much enzymatic protein to sustain the same flux as the less favorable pryophosphate-dependent glycolytic pathway in C. thermocellum; the Embden-Meyerhof-Parnas pathway in E. coli showed intermediate thermodynamic favorability and enzyme demand. The highly reversible fermentation pathway in C. thermocellum requires 10x more protein than the irreversible pathways in Z. mobilis.

Significance

This study provides in vivo evidence that strongly thermodynamically favorable metabolic pathways require significantly lower enzyme concentrations to sustain a given flux than thermodynamically constrained pathways. The insights and quantitative proteomic data generated will serve as a valuable resource for developing constraint-based genome-scale metabolic models.

Khana, D. et al. Thermodynamics shapes the in vivo enzyme burden of glycolytic pathways. mBio, 0, e01837-25. (2025). [DOI:10.1128/mbio.01837-25]
Research Theme
Sustainable Biomass Conversion
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