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


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.

  • Source
    • "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"
    [Show abstract] [Hide abstract]
    ABSTRACT: Modeling cellular metabolism is fundamental for many biotechnological applications, including drug discovery and rational cell factory design. Central carbon metabolism (CCM) is particularly important as it provides the energy and precursors for other biological processes. However, the complex regulation of CCM pathways has still not been fully unraveled and recent studies have shown that CCM is mostly regulated at post-transcriptional levels. In order to better understand the role of allosteric regulation in controlling the metabolic phenotype, we expand the reconstruction of CCM in Escherichia coli with allosteric interactions obtained from relevant databases. This model is used to integrate multi-omics datasets and analyze the coordinated changes in enzyme, metabolite, and flux levels between multiple experimental conditions. We observe cases where allosteric interactions have a major contribution to the metabolic flux changes. Inspired by these results, we develop a constraint-based method (arFBA) for simulation of metabolic flux distributions that accounts for allosteric interactions. This method can be used for systematic prediction of potential allosteric regulation under the given experimental conditions based on experimental data. We show that arFBA allows predicting coordinated flux changes that would not be predicted without considering allosteric regulation. The results reveal the importance of key regulatory metabolites, such as fructose-1,6-bisphosphate, in controlling the metabolic flux. Accounting for allosteric interactions in metabolic reconstructions reveals a hidden topology in metabolic networks, improving our understanding of cellular metabolism and fostering the development of novel simulation methods that account for this type of regulation.
    Full-text · Article · Oct 2015 · Frontiers in Bioengineering and Biotechnology
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite, Trypanosoma brucei. In the mammalian bloodstream, the trypanosome’s metabolism differs significantly from that of its host. For example, the parasite relies exclusively on glycolysis for energy source. Recently, computational and mathematical models of trypanosome metabolism have been generated to assist in understanding the parasite metabolism with the aim of facilitating drug development. Optimisation of these models requires quantitative information, including metabolite concentrations and/or metabolic fluxes that have been hitherto unavailable on a large scale. Here, we have implemented an LC–MS-based method that allows large scale quantification of metabolite levels by using U-13C-labelled E. coli extracts as internal standards. Known amounts of labelled E. coli extract were added into the parasite samples, as well as calibration standards, and used to obtain calibration curves enabling us to convert intensities into concentrations. This method allowed us to reliably quantify the changes of 43 intracellular metabolites and 32 extracellular metabolites in the medium over time. Based on the absolute quantification, we were able to compute consumption and production fluxes. These quantitative data can now be used to optimise computational models of parasite metabolism.
    Full-text · Article · Jul 2015 · Metabolomics
    • "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] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activity, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and GTP regeneration. Magnesium (Mg(2+)), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counter-ion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg(2+) limitation because ATP is the most abundant nucleotide triphosphate in the cell and Mg(2+) is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg(2+) limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg(2+) to indispensable cellular processes. Copyright © 2015. Published by Elsevier Ltd.
    No preview · Article · Jul 2015 · Journal of Molecular Biology
Show more