This study investigates the absolute concentrations of over 100 metabolites in *Escherichia coli* grown aerobically and exponentially with glucose, glycerol, or acetate as carbon sources. The total intracellular metabolite pool is estimated to be approximately 300 mM, with a small number of metabolites dominating the metabolome on a molar basis, particularly glutamate. Most substrate-enzyme pairs have metabolite concentrations exceeding their Michaelis-Menten (K_m) values, except for lower glycolysis, where concentrations are near K_m, indicating near equilibrium. This near-equilibrium state may facilitate efficient flux reversibility under thermodynamic and osmotic constraints. The data and analyses highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data, which is crucial for understanding cellular metabolism and flux directions. The study also examines enzyme saturation, finding that most enzyme active sites are largely saturated, with cofactors like ATP and NAD+ being reliably saturating. The results provide insights into the principles governing metabolite concentrations and enzyme affinities in *E. coli*.This study investigates the absolute concentrations of over 100 metabolites in *Escherichia coli* grown aerobically and exponentially with glucose, glycerol, or acetate as carbon sources. The total intracellular metabolite pool is estimated to be approximately 300 mM, with a small number of metabolites dominating the metabolome on a molar basis, particularly glutamate. Most substrate-enzyme pairs have metabolite concentrations exceeding their Michaelis-Menten (K_m) values, except for lower glycolysis, where concentrations are near K_m, indicating near equilibrium. This near-equilibrium state may facilitate efficient flux reversibility under thermodynamic and osmotic constraints. The data and analyses highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data, which is crucial for understanding cellular metabolism and flux directions. The study also examines enzyme saturation, finding that most enzyme active sites are largely saturated, with cofactors like ATP and NAD+ being reliably saturating. The results provide insights into the principles governing metabolite concentrations and enzyme affinities in *E. coli*.