Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli - PubMed (original) (raw)

Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli

Bryson D Bennett et al. Nat Chem Biol. 2009 Aug.

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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Figure 1

Figure 1. Composition of the measured metabolome

The pie graph shows the molar abundance of different metabolites in glucose-fed cells. Amino acids are shown in dark blue, nucleotides in rust, NAD(P)(H) in yellow, glutathiones in pink, central carbon metabolites in dark green, and all other metabolites in light blue. Abundant metabolites are labeled. Abrevations used: ATP, adenosine-5'-triphosphate; UTP, uridine-5'-triphosphate; GTP, guanosine 5'-triphosphate; dTTP, thymidine 5'-triphosphate; CTP, cytidine-5'-triphosphate; NAD+, nicotinamide adenine dinucleotide; FBP, fructose-1,6-bisphosphate; 6-P-gluconate, 6-phospho-gluconate; Hexose-P, the combined pools of glucose-6-phosphate, glucose-1-phosphate, and fructose-6-phosphate; UDP-N-Ac-Glucosamine, uridine-5'-diphosphate N-acetyl-glucosamine; UDPG, uridine-5'-diphosphate glucose.

Figure 2

Figure 2. Implied enzyme active site saturation

The relationship of metabolite concentration and Km of their consuming enzymes in glucose-grown E. coli. NAD+ is shown as green squares, ATP as yellow squares, NADPH as pinksquares, degradation reactions as blue circles, and reactions in central carbon metabolism (glycolysis, the pentose-phosphate pathway, and the TCA cycle) as orange circles. All other data are shown as grey diamonds. The dark line is the line of unity (where concentration = Km) and the light lines denote a 10-fold deviation from the line of unity.

Figure 3

Figure 3

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