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

Bennett, Bryson D.; Kimball, Elizabeth; Gao, Melissa; Osterhout, Robin; Dien, Stephen J. Van; Rabinowitz, Joshua D.

doi:10.1038/nchembio.186

Cited by 1,643 publications

(1,870 citation statements)

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“…24 Thus, beyond the intrinsic relevance of glucose given its abundance in biological systems and its applications in biotechnological manufacture, studies of proteins in concentrated glucose solutions can in principle help to unveil the effects of small hydrophilic crowders inside cells. Such crowded conditions are very important in shaping life at the molecular level, since they force molecules into unspecific…”

Section: ■ Introductionmentioning

confidence: 99%

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

et al. 2014

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We present herein a thorough description of the effects of high glucose concentrations on the diffusion, hydration and internal dynamics of ubiquitin, as predicted from extensive molecular dynamics simulations on several systems described at fully atomistic level. We observe that the protein acts as a seed that speeds up the natural propensity of glucose to cluster at high concentration; the sugar molecules thus aggregate around the protein trapping it inside a dynamic cage. This process extensively dehydrates the protein surface, restricts the motions of the remaining water molecules, and drags the large-scale, collective motions of protein atoms slowing down the rate of exploration of the conformational space despite only a slight dampening of fast, local dynamics. We discuss how these effects could be relevant to the function of sugars as preservation agents in biological materials, and how crowding by small sticky molecules could modulate proteins across different reaction coordinates inside the cellular cytosol. ■ INTRODUCTIONSugars play roles as agents for the preservation of biological material in nature and in biotechnological manufacture, 1−3 most importantly through their capacity to stabilize proteins against cold and hot denaturation, both in solution and in the solid state. 4−7 Other potential effects of sugars on protein properties have been less explored, but changes in activity, dynamics, and regulation can be expected by analogy to the effects known to be caused by high concentrations of other molecules. Whereas most hydrophilic molecules have the capacity to agglomerate and disrupt water structure, sugars and polyols generally have exceptionally large solubilities, which allows them to strongly dehydrate other molecules and to cluster at very high concentrations forming glassy states. 8−11 Because of these special properties, questions about the effects of sugars on protein properties are intimately related to those revolving around the effects of viscosity, molecular crowding, encapsulation and even vitrification on proteins, all meeting at the crossroads between chemistry, biology, medicine, and applications in the food and pharmacological industries. Within all these closely related fields, the effects of high concentrations of small hydrophilic molecules on protein stability and translational and rotational diffusion have been explored, but studies of their effects on protein hydration and on internal protein dynamics are scarce. 4−7,12−16 More specifically to sugars, reports on the structure and dynamics of sugar-only solutions abound, 8,17−23 but works dealing with proteins in sugar solutions are mostly focused on the stability of the proteins and do not pay much attention to other effects related to hydration, diffusion, and internal mobility. 4−7 We recently reported in a preliminary work that concentrated glucose solutions can perturb protein dynamics by restricting exploration of the conformational space, through a mechanism that presumably involves protein−sugar interac...

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Section: ■ Introductionmentioning

confidence: 99%

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

et al. 2014

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“…We used the software package t efma 24 together with published metabolite concentration data 43 to calculate all TF EFMs in a core metabolic model of E. coli 25 growing on a glucose limited minimal medium. For all unmeasured metabolites in this model we used conservative default concentration ranges between

c_{k}^{truemin} = 10^{- 7} m

and

c_{k}^{truemax} = 1 m

.…”

Section: Methodsmentioning

confidence: 99%

Which sets of elementary flux modes form thermodynamically feasible flux distributions?

et al. 2016

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Elementary flux modes (EFMs) are non‐decomposable steady‐state fluxes through metabolic networks. Every possible flux through a network can be described as a superposition of EFMs. The definition of EFMs is based on the stoichiometry of the network, and it has been shown previously that not all EFMs are thermodynamically feasible. These infeasible EFMs cannot contribute to a biologically meaningful flux distribution. In this work, we show that a set of thermodynamically feasible EFMs need not be thermodynamically consistent. We use first principles of thermodynamics to define the feasibility of a flux distribution and present a method to compute the largest thermodynamically consistent sets (LTCSs) of EFMs. An LTCS contains the maximum number of EFMs that can be combined to form a thermodynamically feasible flux distribution. As a case study we analyze all LTCSs found in Escherichia coli when grown on glucose and show that only one LTCS shows the required phenotypical properties. Using our method, we find that in our E. coli model < 10% of all EFMs are thermodynamically relevant.

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“…Absolute enzyme and metabolite concentrations are fundamental data for the development of reliable metabolic models and rational engineering of industrial microorganisms, as concentrations affect both thermodynamics and dynamics of cellular processes (Bennett et al, 2009;Schomburg, 2009). While many methods have been developed to determine concentrations of cellular components in a sample with high accuracy (Bennett et al, 2008;Tredwell et al, 2011), it is pivotal to measure the intracellular aqueous volume (V in ) precisely, such that absolute concentrations of those cellular components can be reached.…”

Section: Introductionmentioning

confidence: 99%

“…NAD + was chosen as the probe because it can significantly affect the metabolic networks and has been routinely quantified during metabolic data collection (Roca et al, 2003;San et al, 2002). Furthermore, because prokaryotic cells are free of intracellular compartmentalization, the V in data can be readily used for the estimation of absolute concentrations of cellular components (Bennett et al, 2009;Rabinowitz, 2007). This method is relatively simple in terms of attaining accurate V in data because it avoided interferences by other volatile substances.…”

Section: Introductionmentioning

confidence: 99%

An accurate method for estimation of the intracellular aqueous volume of Escherichia coli cells

Wang¹,

Zhou²,

Ji³

et al. 2013

Journal of Microbiological Methods

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

Cited by 1,643 publications

References 47 publications

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

Dissecting the Effects of Concentrated Carbohydrate Solutions on Protein Diffusion, Hydration, and Internal Dynamics

Which sets of elementary flux modes form thermodynamically feasible flux distributions?

An accurate method for estimation of the intracellular aqueous volume of Escherichia coli cells

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