Metabolomics-driven quantitative analysis of ammonia assimilation in E. coli

Despite extensive study of individual enzymes and their organization into pathways, the means by which enzyme networks control metabolite concentrations and fluxes in cells remains incompletely understood. Here, we examine the integrated regulation of central nitrogen metabolism in Escherichia coli through metabolomics and ordinary‐differential‐equation‐based modeling. Metabolome changes triggered by modulating extracellular ammonium centered around two key intermediates in nitrogen assimilation, α‐ketoglutarate and glutamine. Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation. In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes. Combined with covalent modification reactions controlling glutamine synthetase activity, such active‐site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites. Synopsis Although the metabolic pathways converting nutrients to biomass are well known, a quantitative understanding of the relationship between nutrient environment, metabolism, and growth rate is still missing. Furthermore, despite much progress towards quantitatively understanding potential mechanisms for controlling metabolite concentrations and fluxes (as reviewed in Sorribas and Savageau, 1989 ; Heinrich and Schuster, 1996 ; Fell, 1997 ), the most important control mechanisms operating in cells have not been rigorously dissected. This is particularly true for networks containing cycles and branches. Ammonia assimilation in E. coli (Figure 1A ) provides a tractable model network for quantitative analysis of cellular metabolic regulation. This network is composed of two key ammonia assimilating pathways, glutamine synthetase/glutamate synthase (GS/GOGAT) cycle and glutamate dehydrogenase (GDH) pathway, involving three key metabolites (glutamine, glutamate, and α‐ketoglutarate). The enzyme constituents have been explicitly studied biochemically (Miller and Stadtman, 1972 ; Schutt and Holzer, 1972 ; Mantsala and Zalkin, 1976 ; Kustu et al , 1984 ; Alibhai and Villafranca, 1994 ). GS activity is known to be allosterically regulated in response to the intracellular concentrations of glutamine and α‐ketoglutarate (Figure 1B ) (Schutt and H