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.