Genome-scale architecture of small molecule regulatory networks and the fundamental trade-off between regulation and enzymatic activity

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurements and the SMRN to make inferences on the sensitivity of enzymes to their regulators. Generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.P30 CA008748 - NCI NIH HHS; R01 GM121950 - NIGMS NIH HH

Design of stable metabolic networks

In this work, we propose eigenvalue optimization combined with Lyapunov theory concepts to ensure stability of the Embden—Meyerhof–Parnas pathway, the pentosephosphate pathway, the phosphotransferase system and fermentation reactions of Escherichia coli. We address the design of a metabolic network for the maximization of different metabolite production rates. The first case study focuses on serine production, based on a model that consists of 18 differential equations corresponding to dynamic mass balances for extracellular glucose and intracellular metabolites, and thirty kinetic rate expressions. A second case study addresses the design problem to maximize ethanol production, based on a dynamic model that involves mass balancesfor 25 metabolites and 38 kinetic rate equations. The nonlinear optimization problem including stability constraints has been solved with reduced space Successive Quadratic Programming techniques. Numerical results provide useful insights on the stability properties of the studied kinetic models.Fil: Di Maggio, Jimena Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Blanco, Anibal Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Bandoni, Jose Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Díaz, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentin

Metabolite concentrations, fluxes and free energies imply efficient enzyme usage.

In metabolism, available free energy is limited and must be divided across pathway steps to maintain a negative ΔG throughout. For each reaction, ΔG is log proportional both to a concentration ratio (reaction quotient to equilibrium constant) and to a flux ratio (backward to forward flux). Here we use isotope labeling to measure absolute metabolite concentrations and fluxes in Escherichia coli, yeast and a mammalian cell line. We then integrate this information to obtain a unified set of concentrations and ΔG for each organism. In glycolysis, we find that free energy is partitioned so as to mitigate unproductive backward fluxes associated with ΔG near zero. Across metabolism, we observe that absolute metabolite concentrations and ΔG are substantially conserved and that most substrate (but not inhibitor) concentrations exceed the associated enzyme binding site dissociation constant (Km or Ki). The observed conservation of metabolite concentrations is consistent with an evolutionary drive to utilize enzymes efficiently given thermodynamic and osmotic constraints

SBML Reaction Finder: Retrieve and extract specific reactions from the BioModels database

Summary: The SBML Reaction Finder (SRF) application leverages the deep semantic annotations in the BioModels database to provide efficient retrieval and extraction of individual reactions from SBML models. We hope that the SRF will be useful to quantitative modelers who seek to accelerate their modeling efforts by reusing previously published representations of specific chemical reactions.

Availability and Implementation: The SRF is open source, coded in Java, and distributed under the Mozilla Pubic License Version 1.1. Windows, Macintosh and Linux distributions are available for download at 
http://sourceforge.net/projects/sbmlrxnfinder.
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Probing the Details of the Allosteric Inhibition in Phosphofructokinase from Thermus thermophilus

The enzyme phosphofructokinase (PFK,) catalyzes the phosphorylation of fructose-6-phosphate in the glycolysis pathway. Phosphoenolpyruvate (PEP) allosterically inhibits the binding of the substrate fructose-6-phosphate (Fru-6-P) in phosphofructokinase from Thermus thermophilus (TtPFK). The main goal of this study is to have a better understanding about how this allosteric inhibition signal is transmitted and propagated throughout the enzyme. TtPFK is homotetramer with four active sites and four allosteric sites. There are multiple heterotropic interactions between active sites and allosteric sites. The first part of this dissertation is to isolate the four unique heterotropic inhibition interactions in wild type TtPFK. Our data shows the contribution of the four interactions are not the same, and are additive. This result suggests that the traditional two state model, either the concerted or sequential model, is not sufficient to explain the allosteric regulation in TtPFK. Also, the relative contribution of the four interactions in TtPFK is different from BsPFK and EcPFK. The allosteric coupling between Fru-6-P and PEP in TtPFK is much weaker than BsPFK. N59D/A158T/S215H substitutions increase the coupling free energy of TtPFK similar to BsPFK. The second part of this dissertation is to isolate the four interactions in TtPFK N59D/A158T/S215H to see how the substitutions affect the coupling free energy in each of the four interactions. Our data shows that the substitutions of N59D/A158T/S215H can enhance all of the four interactions, but to different extents. 32 A interaction exhibits the biggest increase in coupling free energy and this big increase makes it the second biggest contribution to TtPFK N59D/A158T/S215H. The coupling free energy in the isolated interactions sums to 69.5% ± 1.5% of the total coupling energy in the native tetramer. The discrepancy is likely due to the mutated residues not all interacting within a single subunit. The third part of this dissertation is to use fluorescence phasor to describe the four species, Apo-TtPFK, TtPFK-Fru-6-P, PEP-TtPFK, and PEP-TtPFK-Fru-6-P, involved in the allosteric coupling between Fru-6-P and PEP. TtPFK has a smaller allosteric coupling between PEP and Fru-6-P as compared to other prokaryotic PFKs which makes it easier to form ternary complex. Unique ternary complexes can be detected at specific positions. Our results suggest that residues F140, L313, F165 and V243 may be in an area important for the propagation and transmission of allosteric information in TtPFK. These four residues are in a region that can detect the structural conflict between Fru-6-P binding and PEP binding

Molecular Cloning and Sequencing of the Bacillus Stearothermophilus 6-Phosphofructo-1-Kinase Gene and of a Partial Rabbit Muscle 6-Phosphofructo-1-Kinase Complementary-Dna.

A system is presented for determining the relationships between structure and function in the allosteric enzyme 6-phosphofructo-1-kinase (PFK). The ATP-dependent phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate catalyzed by PFK is the first reaction unique to the glycolytic pathway. The structure-function relationships in PFK can now be addressed by site-specific mutagenesis. This is a method for directly testing hypotheses concerning the functions of individual amino acids in a protein molecule. The main body of this dissertation is composed of three sections. The first two sections describe the development of a system by which the structure-function relationships in Bacillus stearothermophilus PFK (Bs -PFK) can be investigated using the technique of site-specific mutagenesis. The third section describes preliminary efforts made toward establishing a similar system for the PFK from rabbit muscle. (I) Nucleotide sequence of the 6-phosphofructo-1-kinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene: This section describes the cloning and sequencing of the gene encoding Bs -PFK. A significant degree of homology exists when the deduced amino acid sequence of Bs-PFK is compared with the sequences of rabbit muscle PFK or the major PFK from E. coli. (II) High-level expression of Bacillus stearothermophilus 6-phosphofructo-1-kinase in Escherichia coli: This section describes the subcloning of the Bs -PFK gene into a plasmid vector and the high level of Bs -PFK expression which results when this construction is introduced into a PFK null strain of E. coli. This high level of Bs -PFK expression completes the system required for determining the relationships between structure and function in Bs -PFK by site-specific mutagenesis. (III) Molecular cloning and sequencing of a partial cDNA for rabbit muscle 6-phosphofructo-1-kinase: The nucleotide sequence of the cDNA described in this section confirms corresponding portions of the genomic sequence for rabbit muscle PFK. The cloning of the rabbit muscle PFK cDNA fragment represents significant progress toward the long-term goal of using site-specific mutagenesis to determine the structure-function relationships in this allosteric enzyme

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

<p>Abstract</p> <p>Background</p> <p>The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent <it>K</it>_mof the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude.</p> <p>Results</p> <p>We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold.</p> <p>Conclusions</p> <p>Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of <it>K</it>_massociated with kinase enzymes.</p

Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia

10.1371/journal.pone.0056037PLoS ONE82

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases.

The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. Results: We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. Conclusions:Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of Km associated with kinase enzymes

Designing Predictive Mathematical Models for the Metabolic Pathways Associated with Polyhydroxybutyrate Synthesis in \u3ci\u3eEscherichia coli\u3c/i\u3e

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate that has been extensively studied as a potential biodegradable replacement for petrochemically derived plastics. The synthesis pathway of PHB is native to Ralstonia eutropha, but the genes for the PHB pathway have successfully been introduced into Escherichia coli through plasmids such as the pBHR68 plasmid. However, the production of PHB needs to be more cost-effective before it can be commercially produced. A mathematical model for PHB synthesis was developed to identify target genes that could be genetically engineered to increase PHB production. The major metabolic pathways included in the model were glycolysis, acetyl coenzyme A (acetyl-CoA) synthesis, tricarboxylic acid (TCA) cycle, glyoxylate bypass, and PHB synthesis. Each reaction in the selected metabolic pathways was modeled using the kinetic mechanism identified for the associated enzyme. The promoters and transcription factors for each enzyme were incorporated into the model. The model was validated through comparison with other published models and experimental PHB production data. The predictive model identified 16 enzymes as having no effect on PHB production, 5 enzymes with a slight effect on PHB production, and 9 enzymes with large effects on PHB production. Decreasing the substrate affinity of the enzyme citrate synthase resulted in the largest increase in PHB synthesis. The second largest increase was observed from lowering the substrate affinity of glyceraldehyde-3-phosphate dehydrogenase. The predictive model also indicated that increasing the activity of the lac promoter in the pBHR68 plasmid resulted in the largest increase in the rate of PHB production. The predictive model successfully identified two genes and one promoter as targets for genetic engineering to create an optimized strain of E. coli for PHB production. The substrate-binding sites for the genes gltA (citrate synthase) and gapA (glyceraldehyde-3-phosphate dehydrogenase) should be genetically engineered to be less effective at binding the substrates. The lac promoter in the pBHR68 plasmid should be genetically engineered to more closely match the consensus sequence for binding to RNA polymerase. The model predicts that an optimized strain of E. coli for PHB production could be achieved by genetically altering gltA, gapA, and the lac promoter