Thermodynamics shapes the <i>in vivo</i> 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-<i>Zymomonas mobilis</i>, <i>Escherichia coli</i>, and <i>Clostridium thermocellum</i>-which employ distinct glycolytic pathways with varying thermodynamic driving forces. By integrating enzyme concentration data with corresponding <i>in vivo</i> metabolic fluxes and Δ<i>G</i> measurements, we found that the highly favorable Entner-Doudoroff pathway in <i>Z. mobilis</i> requires only one-fourth the amount of enzymatic protein to sustain the same flux as the thermodynamically constrained pyrophosphate-dependent glycolytic pathway in <i>C. thermocellum</i>, with the Embden-Meyerhof-Parnas pathway in <i>E. coli</i> 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 <i>C. thermocellum</i> requires 10-fold more protein to maintain the same flux as the irreversible, forward-driven ethanol fermentation pathway in <i>Z. mobilis</i>. Thus, protein investment across glycolytic pathways reflects differences in their thermodynamic favorability.IMPORTANCECells regulate metabolic fluxes to balance energy production, biosynthesis, and the efficient use of limited resources, including the finite capacity for synthesizing and maintaining metabolic enzymes. Here, we present <i>in vivo</i> evidence that strongly thermodynamically favorable metabolic pathways require significantly fewer enzyme resources to sustain a given flux compared to less thermodynamically favorable pathways. These findings underscore the connection between pathway thermodynamics, resource allocation, and enzyme burden, providing valuable insights for metabolic engineering strategies aimed at optimizing pathways for high flux with minimal protein cost.