Thermodynamics shape the enzyme burden of glycolytic pathways.

Thermodynamically constrained reactions and pathways are hypothesized to impose greater protein demands on cells, requiring higher enzyme amounts to sustain a given flux compared to those with stronger thermodynamics. To test this, we quantified the absolute concentrations of glycolytic enzymes in three bacterial species -, , and - which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding metabolic fluxes and measurements, we found that the highly favorable Entner-Doudoroff (ED) pathway in requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in , with the Embden-Meyerhof-Parnas (EMP) pathway in exhibiting intermediate thermodynamic favorability and enzyme demand. Across all three pathways, early reactions with stronger thermodynamic driving forces generally required lower enzyme investment than later, less favorable steps. Additionally, reflecting differences in glycolytic strategies, the highly reversible ethanol fermentation pathway in requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in . Thus, thermodynamic driving forces constitute a major determinant of the enzyme burden in metabolic pathways.