Here we report systems level analysis of absolute intracellular metabolite concentrations in E. coli
. The analytical approach obtained high sensitivity and specificity via MS/MS while minimizing systematic error by flash-quenching metabolism and including isotope-labeled internal standards throughout. A limitation of the analytical approach was the inability to differentiate free and macromolecule-bound metabolites, as both could be released via organic extraction. As the measured total metabolome concentration of 300 mM (100 million metabolites/cell) greatly exceeded the reported total protein concentration of 7 mM (2.4 million proteins/cell)37
, it is likely that the measured values largely reflect free metabolites. Consistent with this, the dataset was validated as thermodynamically feasible using TMFA. These factors give confidence in the reliability of the measured concentrations.
A striking feature of the observed data was the domination of the metabolome on a molar basis by a small number of compounds, with glutamate comprising over 40% of the total measured intracellular metabolome. Glutamate is the major nitrogen donor in the cell, distributing ~88% of the total nitrogen that ends up in biomass, largely via transamination reactions38
. As transamination reactions have standard free energies near zero, the high concentration of glutamate may be important for driving these reactions forward. Glutamate, however, also has an additional role as the major intracellular counter-ion to potassium39
. The second most abundant metabolite, glutathione, functions as an antioxidant. Thus, the two most abundant “metabolites” each have functions beyond serving as enzyme substrates. For metabolites whose sole role is to serve as enzyme substrates, concentrations were uniformly less than 22 mM, and under 1 mM in 70% of cases. Low concentrations are favorable for avoiding osmotic stress and disadvantageous spontaneous reactions.
The crowded nature of the cytosol, combined with the high costs of protein biosynthesis, favors achieving the metabolic fluxes required for growth with minimal enzyme concentrations, i.e., maximizing flux per enzyme40
. Thus, maintaining substrate concentrations high enough to saturate enzyme active sites should theoretically be beneficial. Consistent with this, most measured metabolites had concentrations that were higher than the Km
of their consuming enzymes. This was particularly true of the ubiquitous cofactors ATP and NAD+
, but also true for measured metabolites more generally. A potential caveat is that the 103 metabolites measured here, and the associated 377 metabolite-enzyme pairs, may not be representative of the entire metabolome, as the measured compounds may be biased towards more abundant ones.
A consequence of maintaining substrate concentrations well above enzyme Km
is relative insensitivity of flux to substrate concentration. Such insensitivity could potentially lead to large swings in metabolite concentrations: flux would not be strongly activated when substrate accumulates, nor terminated when substrate concentration falls. To avoid large swings in metabolite concentrations, flux regulation by competitive inhibition, allostery, covalent modification, or control of enzyme concentrations (e.g., via transcriptional regulation) is accordingly important. Notably, competition for enzyme active sites has the potential to restore sensitivity of flux to substrate concentration, even when substrate is present at substantially above the nominal enzyme Km
. For an irreversible reaction with competitive inhibition, half-maximal reaction velocity occurs at a substrate concentration equal to Km
), where [I] is the concentration of the competitive inhibitor and Ki
is its dissociation constant. Such competition is a reasonable possibility, given the structural similarity of many metabolites and the ubiquitous possibility of enzyme inhibition by its product. Currently, insufficient Ki
values are readily available in literature to systematically analyze the extent of competitive inhibition in E. coli
. We have, however, identified a few cases where active site competition appears to occur. These include glutamate and α-ketoglutarate (23
) competing for the active site of aspartate aminotransferase (with Km
of 0.90 mM and Ki
of 0.15 mM respectively)41
and glutamine (24
), glutamate, and aspartate (25
) competing for the active site of glutamate synthase (with Km
of 0.25 and Ki
of 28.0 and 1.75 mM respectively)42
. Looking forward, the present data set provides the physiologically relevant concentrations of metabolites at which to test competitive and allosteric effects biochemically.
Given the general principle that, for desirable reactions, substrate concentrations are maintained above enzyme Km to avoid “wasting” enzyme active sites, it is notable that substrate concentrations are close to Km for many reactions of central carbon metabolism. We believe that this reflects constraints imposed by the bidirectional nature of central carbon metabolic pathways. One constraint involves the need for fast enzymes (i.e., ones with high kcat) to rapidly release product. For bidirectional reactions, this precludes a very low Km, as fast release of product in one direction implies fast release of substrate in the other (i.e., high Km). Thus, to enable reasonably fast flux in both directions, substrate Km in both directions must be reasonably large.
Others constraints, including thermodynamics, osmotic stress, and harmful side-reactions (like DHAP or glyceradlehyde-3-phosphate (26
) forming the toxic compound methylglyoxal (27
) preclude raising substrate concentrations above the relatively large Km
values required for fast bidirectional catalysis. Because the reactions from FBP to PEP are not strongly forward driven in any of the studied carbon sources, increases in the concentrations of downstream metabolites must be matched by increases in upstream ones to maintain the thermodynamic driving force. Due to the 2:1 stoichiometry between trioses and FBP, a ten-fold increase in a triose like DHAP (desirable to enhance enzyme saturation) would require a 100-fold increase in FBP to avoid changing the pathway thermodynamics. FBP is already the third most abundant compound in glucose-fed E. coli
(15 mM intracellular), therefore such an increase would result in osmotic problems. The 15 mM concentration of FBP may have been evolutionarily selected to optimize the tradeoff between enzyme saturation and osmotic impact.
Consistent with the hypothesis that bidirectional enzymes are less reliably substrate-saturated than unidirectional ones, only four enzymes in central carbon metabolism are saturated with their carbon substrates, and three of these operate in only glycolysis or gluconeogenesis (not both): fructose-bisphosphatase, phosphofructokinase, and fructose-bis-phosphate aldolase A. Some putatively unidirectional enzymes of central carbon metabolism (citrate synthase, α-ketoglutarate dehydrogenase, phosphoenolpyruvate carboxylase) are not substrate saturated, however. Citrate synthase and α-ketoglutarate dehydrogenase sit at metabolic branch points between central carbon metabolism and biosynthesis. Phosphoenolpyruvate carboxylase consumes PEP, which is also used to transport carbon into the cell. Accordingly, the relatively high Km of the central carbon metabolic enzyme may be advantageous for directing flux towards biosynthesis or transport when substrate becomes scarce. Consistent with this, the Km of glutamate synthase for α-ketoglutarate is 17-fold lower than is the Km of glutamate for α-ketoglutarate dehydrogenase. Data for the competing reactions in the other two cases are not available.
A more straightforward example of high enzyme Km being advantageous for flux control arises in degradation pathways. Such pathways enable cells to catabolize end products (e.g., amino acids, nucleosides) that they scavenge from the environment or generate via macromolecule degradation, but have little utility to cells growing exponentially in minimal media. For cells grown on minimal media, we found that Km typically exceeds substrate concentration for degradation enzymes. This provides a double check (in addition to transcriptional regulation) against disadvantageous futile cycling.
These examples highlight an emerging ability to understand the principles underlying the absolute concentrations of metabolites and the affinities of enzymes for their substrates in E. coli. A basic rule is to keep enzymes saturated without letting metabolites build up enough to have osmotic effects. Exceptions arise when the Km values of enzymes that consume the same substrate vary in order to prioritize certain reactions or prevent deleterious ones. For the bidirectional pathways of central carbon metabolism, the need to rapidly release product in both directions and simultaneously conform to thermodynamic constraints prevents substrate concentration from climbing significantly above enzyme Km.