Abstract
Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concentrations in aerobic, exponentially growing Escherichia coli with glucose, glycerol or acetate as the carbon source. The total observed intracellular metabolite pool was approximately 300 mM. A small number of metabolites dominate the metabolome on a molar basis, with glutamate being the most abundant. Metabolite concentration exceeds Km for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the Km of their consuming enzymes and all reactions are near equilibrium. This may facilitate efficient flux reversibility given thermodynamic and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data.
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Acknowledgements
We are indebted to B. Cravatt for the suggestion that metabolite concentrations be compared to the Km of consuming enzymes. We thank the US National Science Foundation (CAREER Award), the Arnold and Mabel Beckman Foundation, the US National Institutes of Health (Center for Quantitative Biology grant P50 GM071508) and the American Heart Association for their financial support.
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B.D.B. performed experiments, analyzed data and wrote the paper. E.H.K. and M.G. performed experiments. R.O. and S.J.V.D. conducted the TMFA. J.D.R. analyzed data and wrote the paper.
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Bennett, B., Kimball, E., Gao, M. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol 5, 593–599 (2009). https://doi.org/10.1038/nchembio.186
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DOI: https://doi.org/10.1038/nchembio.186
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