This study constructed cell-free glycolytic enzyme systems and compared them to their in vivo functions in Escherichia coli. Under in vitro conditions, flux regulation followed enzyme concentrations and kinetics. In E. coli, only one of the isozymes of phosphofructokinase (PfkA) and fructose-bisphosphate aldolase (FbaA) facilitate Embden-Meyerhof-Parnas (EMP) flux, but under in vitro assays, these isozymes were interchangeable. Additionally, in vitro introduction of the Entner-Doudoroff (ED) pathway improved glycolysis rates, while in vivo overexpression of the ED pathway could not capture significant flux unless its phosphotransferase system (PTS) was knocked out. Lastly, in vivo dynamic (13) C-experiments revealed that the labeling order of EMP pathway intermediates was not strictly cascade, indicating intracellular metabolites were not well mixed. These enigmatic observations cannot be fully explained by thermodynamics or substrate level regulations. This article supports the long-time conjecture that EMP enzymes are channeled, and the PTS may be an anchor point to initiate enzyme assemblies. (c) 2018 American Institute of Chemical Engineers AIChE J, 65: 483-490, 2019