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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    • "The lytic tension of Escherichia coli membranes equates to around 250 mmHg applied pressure across the membrane and this can be achieved by a simple rise in the concentrations of cytoplasmic metabolites of around 10 mM [3]. In addition, extant metabolism predominantly generates anions [30] that must be accompanied by either protons generated from metabolism or other cations, which today are principally potassium or sodium ions, though some contribution from magnesium and organic polycations (polyamines) are also evident in today's cytoplasm [30]. The movements of K + and Na + in exchange for protons would be required to stabilise the cytoplasmic pH [31], so channel functions might initially have also evolved for this purpose. "
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    ABSTRACT: Mechanosensitive channels are ubiquitous and highly studied. However, the evolution of the bacterial channels remains enigmatic. It can be argued that mechanosensitivity might be a feature of all membrane proteins with some becoming progressively less sensitive to membrane tension over the course of evolution. Bacteria and archaea exhibit two main classes of channels, MscS and MscL. Present day channels suggest that the evolution of MscL may be highly constrained, whereas MscS has undergone elaboration via gene fusion (and potentially gene fission) events to generate a diversity of channel structures. Some of these channel variants are constrained to a small number of genera or species. Some are only found in higher organisms. Only exceptionally have these diverse channels been investigated in any detail. In this review we consider both the processes that might have led to the evolved complexity but also some of the methods exploiting the explosion of genome sequences to understand (and/or track) their distribution. The role of MscS-related channels in calcium-mediated cell biology events is considered.
    Cell Calcium 12/2014; 57(3). DOI:10.1016/j.ceca.2014.12.011 · 3.51 Impact Factor
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    • "The next step was to target the availability of acetyl-CoA and succinyl-CoA, the two condensation substrates for adipic acid biosynthesis in recombinant E. coli. According to Bennett et al. (2009), intracellular acetyl-CoA and succinyl-CoA concentrations were determined to be 0.61 and 0.23 mM for the aerobically growing cells of E. coli. Analysis of the above adipic acid producers revealed that appreciable levels of acetic acid (1–3 g/L) accumulated in the culture media, while succinic acid did not (data not shown). "
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    Biotechnology and Bioengineering 12/2014; 111(12). DOI:10.1002/bit.25293 · 4.13 Impact Factor
    • "12 h after induction, 25 mL samples were centrifuged (2876 × g, 4 • C, 15 min). Subsequently, pellets were resuspended in quenching buffer (acetonitrile:methanol:H 2 O 4:4:2 with 0.1 M formic acid [33]) at 4 • C and mixed vigorously by vortexing every 3 min while incubating for 10 min on ice, before neutralizing the suspensions with 1 M NH 4 OH. Then, samples were centrifuged again (22,410 × g, 4 • C 10 min). "
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    ChemBioChem 09/2014; 15(13). DOI:10.1002/cbic.201402070 · 3.09 Impact Factor
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