New thermodynamic activity-based approach allows predicting the feasibility of glycolysis

Thermodynamic feasibility analyses help evaluating the feasibility of metabolic pathways. This is an important information used to develop new biotechnological processes and to understand metabolic processes in cells. However, literature standard data are uncertain for most biochemical reactions yielding wrong statements concerning their feasibility. In this article we present activity-based equilibrium constants for all the ten glycolytic reactions, accompanied by the standard reaction data (standard Gibbs energy of reaction and standard enthalpy of reaction). We further developed a thermodynamic activity-based approach that allows to correctly determine the feasibility of glycolysis under different chosen conditions. The results show for the first time that the feasibility of glycolysis can be explained by thermodynamics only if (1) correct standard data are used and if (2) the conditions in the cell at non-equilibrium states are accounted for in the analyses. The results here will help to determine the feasibility of other metabolisms and to understand metabolic processes in cells in the future

Phosphorylation status of pyruvate dehydrogenase in the mousebird Colius striatus undergoing torpor

DATA AVAILABILITY STATEMENT : Data available on request from the authors.Torpor is a heterothermic response that occurs in some animals to reduce metabolic expenditure. The speckled mousebird (Colius striatus) belongs to one of the few avian taxa possessing the capacity for pronounced torpor, entering a hypometabolic state with concomitant decreases in body temperature in response to reduced food access or elevated thermoregulatory energy requirements. The pyruvate dehydrogenase complex (PDC) is a crucial site regulating metabolism by bridging glycolysis and the Krebs cycle. Three highly conserved phosphorylation sites are found within the E1 enzyme of the complex that inhibit PDC activity and reduce the flow of carbohydrate substrates into the mitochondria. The current study demonstrates a marked increase in S232 phosphorylation during torpor in liver, heart, and skeletal muscle of C. striatus. The increase in S232 phosphorylation during torpor was particularly notable in skeletal muscle where levels were ~49-fold higher in torpid birds compared to controls. This was in contrast to the other two phosphorylation sites (S293 and S300) which remained consistently phosphorylated regardless of tissue. The relevant PDH kinase (PDHK1) known to phosphorylate S232 was found to be substantially upregulated (~5-fold change) in the muscle during torpor as well as increasing moderately in the liver (~2.2-fold increase). Additionally, in the heart, a slight (~23%) decrease in total PDH levels was noted. Taken together the phosphorylation changes in PDH suggest that inhibition of the complex is a common feature across several tissues in the mousebird during torpor and that this regulation is mediated at a specific residue.Natural Sciences and Engineering Research Council of Canada and National Research Foundation of South Africa.http://wileyonlinelibrary.com/journal/jezhj2023Zoology and Entomolog

Consistent Estimation of Gibbs Energy Using Component Contributions

Standard Gibbs energies of reactions are increasingly being used in metabolic modeling for applying thermodynamic constraints on reaction rates, metabolite concentrations and kinetic parameters. The increasing scope and diversity of metabolic models has led scientists to look for genome-scale solutions that can estimate the standard Gibbs energy of all the reactions in metabolism. Group contribution methods greatly increase coverage, albeit at the price of decreased precision. We present here a way to combine the estimations of group contribution with the more accurate reactant contributions by decomposing each reaction into two parts and applying one of the methods on each of them. This method gives priority to the reactant contributions over group contributions while guaranteeing that all estimations will be consistent, i.e. will not violate the first law of thermodynamics. We show that there is a significant increase in the accuracy of our estimations compared to standard group contribution. Specifically, our cross-validation results show an 80% reduction in the median absolute residual for reactions that can be derived by reactant contributions only. We provide the full framework and source code for deriving estimates of standard reaction Gibbs energy, as well as confidence intervals, and believe this will facilitate the wide use of thermodynamic data for a better understanding of metabolism

Which way does the citric acid cycle turn during hypoxia? The critical role of alpha-ketoglutarate dehydrogenase complex

The citric acid cycle forms a major metabolic hub and as such it is involved in many disease states involving energetic imbalance. In spite of the fact that it is being branded as a "cycle", during hypoxia, when the electron transport chain does not oxidize reducing equivalents, segments of this metabolic pathway remain operational but exhibit opposing directionalities. This serves the purpose of harnessing high-energy phosphates through matrix substrate-level phosphorylation in the absence of oxidative phosphorylation. In this Mini-Review, these segments are appraised, pointing to the critical importance of the alpha-ketoglutarate dehydrogenase complex dictating their directionalities. (c) 2013 Wiley Periodicals, Inc

Thermodynamically-Constrained Computational Modeling of Lung Tissue Bioenergetics and the Effect of Hyperoxia-Induced Acute Lung Injury

Altered lung tissue bioenergetics is an important and early step in the pathogenesis of acute lung injury (ALI), one of the most common causes of admission to medical ICUs. A wealth of information exists regarding the effect of ALI on specific mitochondrial and cytosolic processes in isolated mitochondria, cultured endothelial cell, and intact lungs. However, the interdependence of lung cellular processes makes it difficult to quantify the impact of a change in a single or multiple cellular process(es) on overall lung tissue bioenergetics. Integrating bioenergetics data from isolated mitochondria and intact lung is necessary for determining the functional significance of targeting a specific cellular process for prognostic and/or therapeutic purposes. Thus, the main objective of my dissertation was to develop and validate comprehensive, thermodynamically-constrained models of mitochondrial and lung tissue bioenergetics, and to use these models to predict the impact of ALI-induced changes in mitochondrial and cytosolic processes on lung tissue bioenergetics. For Aim 1, I developed an integrated model of the bioenergetics of mitochondria isolated from rat lungs, which incorporates the major biochemical reactions and transport processes in lung mitochondria. The model was validated by assessing its ability to predict experimental data not used for parameter estimation. The model provides important insights into the bioenergetics of lung mitochondria and how they differ from those of mitochondria from other organs. For Aim 2, I developed and validated an integrated computational model of lung tissue bioenergetics. The model expanded the computational model developed under Aim 1 by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. The model was then used to gain novel insights into how lung tissue glycolytic rate is regulated by exogenous substrates, and assess differences in the bioenergetics of isolated mitochondria isolated from lung tissue and those of mitochondria in intact lungs. For Aim 3, the models developed under Aims 1 and 2 were used to quantify the impact of previously measured changes in specific mitochondrial processes in a rat model of clinical ALI on lung mitochondrial and tissue bioenergetics. To the best of our knowledge, the two computational models are the first for lung mitochondrial and tissue bioenergetics. These models provide a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics and the pathogenesis of ALI, and for identifying potential therapeutic targets for ALI

Comparação de métodos computacionais para estimar a energia livre de Gibbs em reações bioquímicas

Dissertação de mestrado em Biofísica e BionanossistemasA energia livre de Gibbs, por indicar o saldo energético de uma reação química ou bioquímica, fornece indicações acerca da direção e da reversibilidade que essa reação e, em última instância, uma via metabólica completa, podem tomar. Estas informações podem ser de uma utilidade crucial na concepção de processos bioquímicos da maior relevância quer em investigação científica, quer ao nível de aplicações inovadoras nas mais variadas áreas da biotecnologia. No entanto, a disponibilidade de dados termodinâmicos concretos e, em especial, sobre a variação da energia livre de Gibbs, em reações do metabolismo celular é escassa e, frequentemente, incompleta. Ademais, os constrangimentos práticos da sua obtenção pela via experimental impedem a avaliação termodinâmica de grande número de reacções. Os métodos estimativos são por isso uma opção atrativa, tendo já sido publicados por vários autores diversos trabalhos que visam o desenvolvimento de formas alternativas de avaliar a variação da energia livre de Gibbs de um elevado número de reacções num curto espaço de tempo. Os esforços mais recentes e promissores neste sentido procuram combinar, de uma forma completa e consistente, os dados obtidos pela via experimental com diferentes abordagens teóricas sobre a previsibilidade da termodinâmica ao nível de reações bioquímicas. Nesta dissertação, colocam-se em destaque dois métodos em particular. O primeiro, Component Contribution, utiliza dados provenientes de trabalhos experimentais, aliados a outras informações teóricas e a um princípio assente na decomposição das espécies químicas envolvidas em vários grupos funcionais. O segundo, por sua vez, baseia-se na ideia de que os dados experimentais disponíveis na literatura podem ser aplicados segundo uma lógica de semelhanças estruturais e transformacionais para inferir dados acerca de reações para as quais os parâmetros termodinâmicos são ainda desconhecidos. Na impossibilidade de aplicar diretamente este segundo método nos seus moldes originais, neste trabalho optou-se por desenvolver de raíz um programa baseado nos mesmos princípios teóricos. São ainda tecidas considerações sobre o âmbito de aplicabilidade de cada um dos métodos abordados e avaliada a capacidade de um poder complementar o outro, numa ferramenta mais completa e mais robusta.For its ability to denote the energetic balance of a chemical or biochemical reaction, Gibbs free energy provides information about the direction and reversibility that it, or even a complete metabolic pathway, can take. Such information may be of great value for designing biochemical processes of huge relevance for both scientific research and innovative applications in the most diverse fields of biotechnology. However, availability of specific data about the thermodynamics – and especially about the Gibbs free energy – of metabolic reactions is limited and often incomplete. Moreover, practical constraints involved in their experimental measurement prevent ascertaining thermodynamical parameters for a large number of reactions. Estimation methods pose therefore an attractive option, and several works have been published envisioning the development of alternative ways for evaluating the change in Gibbs free energy for large sets of reactions in a short time. The most recent and promising efforts look for consistently combining experimental data and different theoretical approaches on predicting thermodynamics in biochemical reactions. This thesis focuses on two methods. The first one, Component Contribution, uses data from experimental works together with other theoretical information and the idea that chemical species taking part in a reaction can be decomposed into several functional groups. The second one relies on the principle that the available experimental data can be used for searching structural and transformational similarities for inferring data for reactions with unknown thermodynamic parameters. Although the second method could not be implemented in its original formulation, another method was developed from scratch during this work, using the same theoretical principles employed in the original one. The practical applicability of each method was evaluated, as well as the possibility of their implementation in a complementary manner

Development and application of a method for quantitative metabolome analysis of various production strains

Im Rahmen der Dissertation wurde eine Methode zur Quantifizierung der Metabolite des zentralen Kohlenstoffwechsels von Mikroorganismen entwickelt. Die Methode wurde genutzt um das Metabolom verschiedenster Produktionsstämme im nanomolaren Bereich zu analysieren. Bei der Analyse der Daten wurden Ergebnisse aus Metabolom- und Fluxomforschung kombiniert, um einen ganzheitlichen Ansatz zu schaffen. Auf diese Weise konnte unter anderem der Einfluss verschiedener Kultiverungsverfahren auf das Energielevel von E. coli untersucht werden. Weitere Messungen untersuchten den Einfluss von genetischen Veränderungen, Stress und unterschiedlichen C-Quellen auf den zentralen Kohlenstoffwechsel von weiteren Mikroorganismen.The present work describes the development of a analytical method to quantify the metabolites of the central carbon metabolism of microorganisms. The method was used to analyze the metabolome of various production strains in the nanomolar range. The combination of metabolome and fluxome data allowed a wholistic analysis of the measured data. The apporach was used to analyze the influence of different cultivation modes on the adenylate energy charge of E. coli. Furthermore, the influence of genetic modifications, stress or different carbon sources on the central carbon metabolism of other production strains was analyzed by applcation of the developed method