The study quantifies over 100 metabolite concentrations in aerobic, exponentially growing *Escherichia coli* using liquid chromatography-tandem mass spectrometry. The total intracellular metabolite pool is approximately 300 mM, with glutamate being the most abundant. Most substrate-enzyme pairs have metabolite concentrations exceeding their respective $ K_m $ values, except in lower glycolysis, where concentrations are near $ K_m $ and reactions are near equilibrium. This suggests efficient flux reversibility under thermodynamic and osmotic constraints. The data highlight the ability to identify metabolic principles from absolute metabolite concentration data. Absolute metabolite concentrations are critical for understanding metabolic dynamics, enabling the direct determination of flux and providing insights into enzyme saturation levels. These factors are essential for modeling metabolic dynamics. The study also shows that many enzymes are highly saturated, with substrate concentrations far exceeding $ K_m $ values, indicating that enzyme active sites are largely filled. This has implications for reaction rates and sensitivity to substrate, product, and competitive inhibitor concentrations. The study also examines the thermodynamics of metabolic flux in *E. coli* using thermodynamic metabolic flux analysis (TMFA). It finds that most reactions are strongly forward driven, with $ \Delta G > 10 $ kJ/mol in many cases. The availability of absolute concentration data allows for the determination of feasible flux directions, with most reactions being thermodynamically favorable. However, some reactions, such as those involved in glycerol assimilation, require specific enzyme activities to be feasible. The study also assesses enzyme saturation by comparing metabolite concentrations to $ K_m $ values from the BRENDA database. It finds that many enzymes are highly saturated, with substrate concentrations far exceeding $ K_m $ values. This suggests that enzyme active sites are largely filled, which may be beneficial for maintaining metabolic flux. However, some enzymes, particularly those involved in nucleotide, nucleoside, nucleobase, and amino acid degradation, are not saturated, indicating that their substrate concentrations are lower than $ K_m $ values. The study highlights the importance of absolute metabolite concentrations in understanding metabolic principles and enzyme regulation. It also shows that the high concentration of glutamate may be important for driving transamination reactions, which have near-zero standard free energies. Glutamate also serves as a major intracellular counter-ion to potassium, highlighting its multifunctional role. The study also shows that the high concentration of ATP and $ NAD^+ $ may be important for maintaining enzyme activity, as their concentrations typically exceed $ K_m $ values by more than 10-fold. The study also discusses the implications of maintaining substrate concentrations well above enzyme $ K_m $ values. This can lead to relative insensitivity of flux to substrate concentration, which could potentially lead to large swings in metabolite concentrations. To avoid this, flux regulation through competitive inhibition, allostery, covalent modification, or control ofThe study quantifies over 100 metabolite concentrations in aerobic, exponentially growing *Escherichia coli* using liquid chromatography-tandem mass spectrometry. The total intracellular metabolite pool is approximately 300 mM, with glutamate being the most abundant. Most substrate-enzyme pairs have metabolite concentrations exceeding their respective $ K_m $ values, except in lower glycolysis, where concentrations are near $ K_m $ and reactions are near equilibrium. This suggests efficient flux reversibility under thermodynamic and osmotic constraints. The data highlight the ability to identify metabolic principles from absolute metabolite concentration data. Absolute metabolite concentrations are critical for understanding metabolic dynamics, enabling the direct determination of flux and providing insights into enzyme saturation levels. These factors are essential for modeling metabolic dynamics. The study also shows that many enzymes are highly saturated, with substrate concentrations far exceeding $ K_m $ values, indicating that enzyme active sites are largely filled. This has implications for reaction rates and sensitivity to substrate, product, and competitive inhibitor concentrations. The study also examines the thermodynamics of metabolic flux in *E. coli* using thermodynamic metabolic flux analysis (TMFA). It finds that most reactions are strongly forward driven, with $ \Delta G > 10 $ kJ/mol in many cases. The availability of absolute concentration data allows for the determination of feasible flux directions, with most reactions being thermodynamically favorable. However, some reactions, such as those involved in glycerol assimilation, require specific enzyme activities to be feasible. The study also assesses enzyme saturation by comparing metabolite concentrations to $ K_m $ values from the BRENDA database. It finds that many enzymes are highly saturated, with substrate concentrations far exceeding $ K_m $ values. This suggests that enzyme active sites are largely filled, which may be beneficial for maintaining metabolic flux. However, some enzymes, particularly those involved in nucleotide, nucleoside, nucleobase, and amino acid degradation, are not saturated, indicating that their substrate concentrations are lower than $ K_m $ values. The study highlights the importance of absolute metabolite concentrations in understanding metabolic principles and enzyme regulation. It also shows that the high concentration of glutamate may be important for driving transamination reactions, which have near-zero standard free energies. Glutamate also serves as a major intracellular counter-ion to potassium, highlighting its multifunctional role. The study also shows that the high concentration of ATP and $ NAD^+ $ may be important for maintaining enzyme activity, as their concentrations typically exceed $ K_m $ values by more than 10-fold. The study also discusses the implications of maintaining substrate concentrations well above enzyme $ K_m $ values. This can lead to relative insensitivity of flux to substrate concentration, which could potentially lead to large swings in metabolite concentrations. To avoid this, flux regulation through competitive inhibition, allostery, covalent modification, or control of