Article

Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia coli

Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA.
Nature Chemical Biology (Impact Factor: 13). 07/2009; 5(8):593-9. DOI: 10.1038/nchembio.186
Source: PubMed

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 K(m) for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the K(m) 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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    • "In fact , metabolite measurements in E . coli and S . cerevisiae have shown that most enzymes in central carbon metabolism are not saturated , with substrate levels being close to their respective K M values ( Bennett et al . , 2009 ; Fendt et al . , 2010 ) . A recent study in B . subtilis showed that transcriptional regulation is insuffi - cient to explain the observed flux change for growth in differ - ent carbon sources ( Chubukov et al . , 2013 ) . Interestingly , the authors observed that the changes in substrate concentrations were also insufficient to explai"
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    • "However, since the availability of commercial U-13 C-labelled isotopomers is limited and they are often prohibitively expensive, in vivo synthesis of U-13 C-labelled compounds is required using suitable microorganisms grown in U-13 C-labelled glucoselimited culture media. Absolute intracellular concentrations of metabolites in a sample can then be calculated by adding known amounts of U-13 C-labelled cell extract prior to the extraction procedure (Bennett et al. 2009; Mashego et al. 2004). The most suitable method for this isotope ratiobased MS (IR-MS) technique is to use the same organism for generating U-13 C-labelled cell extracts which can cover all of intracellular metabolites for absolute quantification (Mashego et al. 2004; Wu et al. 2005). "
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    • "N/A. ~2 mM is required for maximum activity in vitro [103] 1.2–12 mM [102] ATP 9.6 mM (glucose-fed E. coli [104]) 3 8 –80 μM [105] [106] N9 mM ADP 0.55 mM (glucose-fed E. coli [104]) 300 μM [106] b0.2 mM AMP 0.28 mM (glucose-fed E. coli [104]) 1 0 m M [106] N/A UTP 8.3 mM (glucose-fed E. coli [104]) 6 9 μM [107] N8 mM GTP 4.9 mM (glucose-fed E. coli [104]) Similar to that of ATP [106] N4.5 mM CTP 2.7 mM (glucose-fed E. coli [104]) Similar to that of ATP [106] N2.5 mM PP i (inorganic pyrophosphate) from ~0.2 mM [108] to 0.5 mM [109] ~24 μM [107] "
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