109 research outputs found
Detailed Enzyme Kinetics in Terms of Biochemical Species: Study of Citrate Synthase
The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data. This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme
anNET: a tool for network-embedded thermodynamic analysis of quantitative metabolome data
Background: Compared to other omics techniques, quantitative metabolomics is still at its infancy. Complex sample preparation and analytical procedures render exact quantification extremely difficult. Furthermore, not only the actual measurement but also the subsequent interpretation of quantitative metabolome data to obtain mechanistic insights is still lacking behind the current expectations. Recently, the method of network-embedded thermodynamic (NET) analysis was introduced to address some of these open issues. Building upon principles of thermodynamics, this method allows for a quality check of measured metabolite concentrations and enables to spot metabolic reactions where active regulation potentially controls metabolic flux. So far, however, widespread application of NET analysis in metabolomics labs was hindered by the absence of suitable software.
Results: We have developed in Matlab a generalized software called 'anNET' that affords a user-friendly implementation of the NET analysis algorithm. anNET supports the analysis of any metabolic network for which a stoichiometric model can be compiled. The model size can span from a single reaction to a complete genome-wide network reconstruction including compartments. anNET can (i) test quantitative data sets for thermodynamic consistency, (ii) predict metabolite concentrations beyond the actually measured data, (iii) identify putative sites of active regulation in the metabolic reaction network, and (iv) help in localizing errors in data sets that were found to be thermodynamically infeasible. We demonstrate the application of anNET with three published Escherichia coli metabolome data sets.
Conclusion: Our user-friendly and generalized implementation of the NET analysis method in the software anNET allows users to rapidly integrate quantitative metabolome data obtained from virtually any organism. We envision that use of anNET in labs working on quantitative metabolomics will provide the systems biology and metabolic engineering communities with a mean to proof the quality of metabolome data sets and with all further benefits of the NET analysis approach.
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Molybdenum and Phosphorus Interact to Constrain Asymbiotic Nitrogen Fixation in Tropical Forests
Biological di-nitrogen fixation (N2) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N2 fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N2 conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N2 fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N2 fixation. Fixation is uniformly favored in surface organic soil horizons - a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N2 fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought
A scalable algorithm to explore the Gibbs energy landscape of genome-scale metabolic networks
The integration of various types of genomic data into predictive models of
biological networks is one of the main challenges currently faced by
computational biology. Constraint-based models in particular play a key role in
the attempt to obtain a quantitative understanding of cellular metabolism at
genome scale. In essence, their goal is to frame the metabolic capabilities of
an organism based on minimal assumptions that describe the steady states of the
underlying reaction network via suitable stoichiometric constraints,
specifically mass balance and energy balance (i.e. thermodynamic feasibility).
The implementation of these requirements to generate viable configurations of
reaction fluxes and/or to test given flux profiles for thermodynamic
feasibility can however prove to be computationally intensive. We propose here
a fast and scalable stoichiometry-based method to explore the Gibbs energy
landscape of a biochemical network at steady state. The method is applied to
the problem of reconstructing the Gibbs energy landscape underlying metabolic
activity in the human red blood cell, and to that of identifying and removing
thermodynamically infeasible reaction cycles in the Escherichia coli metabolic
network (iAF1260). In the former case, we produce consistent predictions for
chemical potentials (or log-concentrations) of intracellular metabolites; in
the latter, we identify a restricted set of loops (23 in total) in the
periplasmic and cytoplasmic core as the origin of thermodynamic infeasibility
in a large sample () of flux configurations generated randomly and
compatibly with the prior information available on reaction reversibility.Comment: 11 pages, 6 figures, 1 table; for associated supporting material see
http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.100256
In Silico Evidence for Gluconeogenesis from Fatty Acids in Humans
The question whether fatty acids can be converted into glucose in humans has a long standing tradition in biochemistry, and the expected answer is “No”. Using recent advances in Systems Biology in the form of large-scale metabolic reconstructions, we reassessed this question by performing a global investigation of a genome-scale human metabolic network, which had been reconstructed on the basis of experimental results. By elementary flux pattern analysis, we found numerous pathways on which gluconeogenesis from fatty acids is feasible in humans. On these pathways, four moles of acetyl-CoA are converted into one mole of glucose and two moles of CO2. Analyzing the detected pathways in detail we found that their energetic requirements potentially limit their capacity. This study has many other biochemical implications: effect of starvation, sports physiology, practically carbohydrate-free diets of inuit, as well as survival of hibernating animals and embryos of egg-laying animals. Moreover, the energetic loss associated to the usage of gluconeogenesis from fatty acids can help explain the efficiency of carbohydrate reduced and ketogenic diets such as the Atkins diet
Abiotic ammonium formation in the presence of Ni-Fe metals and alloys and its implications for the Hadean nitrogen cycle
Experiments with dinitrogen-, nitrite-, nitrate-containing solutions were conducted without headspace in Ti reactors (200°C), borosilicate septum bottles (70°C) and HDPE tubes (22°C) in the presence of Fe and Ni metal, awaruite (Ni80Fe20) and tetrataenite (Ni50Fe50). In general, metals used in this investigation were more reactive than alloys toward all investigated nitrogen species. Nitrite and nitrate were converted to ammonium more rapidly than dinitrogen, and the reduction process had a strong temperature dependence. We concluded from our experimental observations that Hadean submarine hydrothermal systems could have supplied significant quantities of ammonium for reactions that are generally associated with prebiotic synthesis, especially in localized environments. Several natural meteorites (octahedrites) were found to contain up to 22 ppm Ntot. While the oxidation state of N in the octahedrites was not determined, XPS analysis of metals and alloys used in the study shows that N is likely present as nitride (N3-). This observation may have implications toward the Hadean environment, since, terrestrial (e.g., oceanic) ammonium production may have been supplemented by reduced nitrogen delivered by metal-rich meteorites. This notion is based on the fact that nitrogen dissolves into metallic melts
On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.
Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life
Lower glycolysis carries a higher flux than any biochemically possible alternative
The universality of many pathways of core metabolism suggests a strong role
for evolutionary selection, but it remains unclear whether existing pathways
have been selected from a large or small set of biochemical possibilities. To
address this question, we construct "in silico" all possible biochemically
feasible alternatives to the trunk pathway of glycolysis and gluconeogenesis,
one of the most highly conserved pathways in metabolism. We show that, even
though a large number of alternative pathways exist, the alternatives carry
lower flux than the real pathway under typical physiological conditions.
Alternative pathways that could potentially carry higher flux often lead to
infeasible intermediate metabolite concentrations. We also find that if
physiological conditions were different, different pathways could outperform
those found in nature. Our results demonstrate how the rules of biochemistry
restrict the alternatives that are open to evolution, and suggest that the
existing trunk pathway of glycolysis and gluconeogenesis represents a maximal
flux solution.Comment: 9 pages, 4 figure
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