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D A Fell

Publications and source records attributed to D A Fell.

At least 19 recordsLinked to original sources
PubMed2002

Reaction routes in biochemical reaction systems: algebraic properties, validated calculation procedure and example from nucleotide metabolism.

Elementary flux modes (direct reaction routes) are minimal sets of enzymes that can operate at steady state, with all irreversible reactions used in the appropriate direction. They can be interpreted as component pathways of a (bio)chemical reaction network. Here, two different definitions of elementary modes are given and their equivalence is proved. Several algebraic properties of elementary modes are then presented and proved. This concerns, amongst other features, the minimal number of enzymes of the network not used in an elementary mode and the situations where irreversible reactions are replaced by reversible ones. Based on these properties, a refined algorithm is presented, and it is formally proved that this algorithm will exclusively generate all the elementary flux modes of an arbitrary network containing reversible or irreversible reactions or both. The algorithm is illustrated by a biochemical example relevant in nucleotide metabolism. The computer implementation in two different programming languages is discussed.

Adenine Nucleotides↗
PubMed2001

The small world inside large metabolic networks.

The metabolic network of the catabolic, energy and biosynthetic metabolism of Escherichia coli is a paradigmatic case for the large genetic and metabolic networks that functional genomics efforts are beginning to elucidate. To analyse the structure of previously unknown networks involving hundreds or thousands of components by simple visual inspection is impossible, and quantitative approaches are needed to analyse them. We have undertaken a graph theoretical analysis of the E. coli metabolic network and find that this network is a small-world graph, a type of graph distinct from both regular and random networks and observed in a variety of seemingly unrelated areas, such as friendship networks in sociology, the structure of electrical power grids, and the nervous system of Caenorhabditis elegans. Moreover, the connectivity of the metabolites follows a power law, another unusual but by no means rare statistical distribution. This provides an objective criterion for the centrality of the tricarboxylic acid cycle to metabolism. The small-world architecture may serve to minimize transition times between metabolic states, and contains evidence about the evolutionary history of metabolism.

Escherichia coli↗
PubMed2001

An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.

We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

Aspartate Kinase↗
PubMed2001

Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics.

We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM.

Adenosine Triphosphate↗
PubMed2001

Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach.

A computer simulation of the threonine-synthesis pathway in Escherichia coli Tir-8 has been developed based on our previous measurements of the kinetics of the pathway enzymes under near-physiological conditions. The model successfully simulates the main features of the time courses of threonine synthesis previously observed in a cell-free extract without alteration of the experimentally determined parameters, although improved quantitative fits can be obtained with small parameter adjustments. At the concentrations of enzymes, precursors and products present in cells, the model predicts a threonine-synthesis flux close to that required to support cell growth. Furthermore, the first two enzymes operate close to equilibrium, providing an example of a near-equilibrium feedback-inhibited enzyme. The predicted flux control coefficients of the pathway enzymes under physiological conditions show that the control of flux is shared between the first three enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase, with no single activity dominating the control. The response of the model to the external metabolites shows that the sharing of control between the three enzymes holds across a wide range of conditions, but that the pathway flux is sensitive to the aspartate concentration. When the model was embedded in a larger model to simulate the variable demands for threonine at different growth rates, it showed the accumulation of free threonine that is typical of the Tir-8 strain at low growth rates. At low growth rates, the control of threonine flux remains largely with the pathway enzymes. As an example of the predictive power of the model, we studied the consequences of over-expressing different enzymes in the pathway.

Aspartate Kinase↗
PubMed2001

Beyond genomics.

Explore the source record for details and available documents.

Galactose↗
PubMed2001

Computer modelling and experimental evidence for two steady states in the photosynthetic Calvin cycle.

We present observations of photosynthetic carbon dioxide assimilation, and leaf starch content from genetically modified tobacco (Nicotiana tabacum) plants in which the activity of the Calvin cycle enzyme, sedoheptulose-1,7-bisphosphatase, is reduced by an antisense construct. The measurements were made on leaves of varying ages and used to calculate the flux control coefficients of sedoheptulose-1,7-bisphosphatase over photosynthetic assimilation and starch synthesis. These calculations suggest that control coefficients for both are negative in young leaves, and positive in mature leaves. This behaviour is compared to control coefficients obtained from a detailed computer model of the Calvin cycle. The comparison demonstrates that the experimental observations are consistent with bistable behaviour exhibited by the model, and provides the first experimental evidence that such behaviour in the Calvin cycle occurs in vivo as well as in silico.

Carbon Dioxide↗
PubMed2000

Differential feedback regulation of the MAPK cascade underlies the quantitative differences in EGF and NGF signalling in PC12 cells.

Although epidermal growth factor (EGF) induces transient activation of Ras and the mitogen-activated protein kinase (MAPK) cascade in PC12 cells, whereas nerve growth factor (NGF) stimulates sustained activation, the basis for these contrasting responses is not known. We have developed a computer simulation of EGF-induced MAPK cascade activation, which provides quantitative evidence that feedback inhibition of the MAPK cascade is the most important factor in determining the duration of cascade activation. Hence, we propose that the observed quantitative differences in EGF and NGF signalling can be accounted for by differential feedback regulation of the MAPK cascade.

Animals↗
PubMed2000

A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks.

A set of linear pathways often does not capture the full range of behaviors of a metabolic network. The concept of 'elementary flux modes' provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. We have used this concept to analyze the interplay between the pentose phosphate pathway (PPP) and glycolysis. The set of elementary modes for this system involves conventional glycolysis, a futile cycle, all the modes of PPP function described in biochemistry textbooks, and additional modes that are a priori equally entitled to pathway status. Applications include maximizing product yield in amino acid and antibiotic synthesis, reconstruction and consistency checks of metabolism from genome data, analysis of enzyme deficiencies, and drug target identification in metabolic networks.

Algorithms↗
PubMed2000

Modelling photosynthesis and its control.

The dynamic and steady-state behaviour of a computer simulation of the Calvin cycle reactions of the chloroplast, including starch synthesis and degradation, and triose phosphate export have been investigated. A major difference compared with previous models is that none of the reversible reactions are assumed to be at equilibrium. The model can exhibit alternate steady states of low or high carbon assimilation flux, with hysteresis in the transitions between the steady states induced by environmental factors such as phosphate and light intensity. The enzymes which have the greatest influence on the flux have been investigated by calculation of their flux control coefficients. Different patterns of control are exhibited over the assimilation flux, the flux to starch and the flux to cytosolic triose phosphate. The assimilation flux is mostly sensitive to sedoheptulose bisphosphatase and Rubisco, with the exact distribution depending on their relative activities. Other enzymes, particularly the triose phosphate translocator, become more influential when other fluxes are considered. These results are shown to be broadly consistent with observations on transgenic plants.

Computer Simulation↗
PubMed1999

Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering.

Rational metabolic engineering requires powerful theoretical methods such as pathway analysis, in which the topology of metabolic networks is considered. All metabolic capabilities in steady states are composed of elementary flux modes, which are minimal sets of enzymes that can each generate valid steady states. The modes of the fructose-2,6-bisphosphate cycle, the combined tricarboxylic-acid-glyoxylate-shunt system and tryptophan synthesis are used here for illustration. This approach can be used for many biotechnological applications such as increasing the yield of a product, channelling a product into desired pathways and in functional reconstruction from genomic data.

Biochemistry↗
PubMed1998

A control analysis exploration of the role of ATP utilisation in glycolytic-flux control and glycolytic-metabolite-concentration regulation.

A theoretical metabolic-control-analysis approach has been used to study aspects of glycolytic-flux control and carbon-metabolite regulation, particularly the role of ATP demand (ATPase), in order to determine what general features of the regulation of energy metabolism would be consistent with good carbon-metabolite homeostasis in the face of large changes in carbon flux. On the basis of a semi-quantitative control-analysis model, incorporating estimates of substrate, product and effector actions on the enzymes, the experimentally observed characteristics of glycolytic-flux changes prove to impose constraints on the feasible ranges of these estimates. This leads to the identification of several features of energy metabolism, each of which is necessary but not sufficient to explain the observations; although most of these have been advocated previously (such as AMP activation of phosphofructokinase (PFK), ADP inhibition of ATPase and the role of energy charge or ATP/ADP ratio), our analysis allows their relative importance to be assessed. In the model, the distribution of flux control depends primarily on ADP inhibition of ATPase, and on the activation of PFK by AMP; increase in ADP inhibition of ATPase increases the control on PFK; increase in AMP activation of PFK increases control on ATPase. PFK exerts greater flux control than does ATPase over approximately 50% of the ranges (parameter space) studied, but its control is sufficiently high to achieve sizeable flux increases over less than 20% of the space. Furthermore, control by alteration in PFK activity is shown to result in poor glycolytic metabolite homeostasis over the entire parameter space studied. However, over a large proportion of the parameter space, control by activation of ATPase can lead to large flux changes, i.e. high flux control, coupled with excellent glycolytic-metabolite homeostasis, similar to that observed in working muscle. As well as altering the relative degrees of flux control invested in PFK and ATPase, ADP inhibition of ATPase and AMP activation of PFK have pronounced effects on the homeostatic properties of the system. Stronger ADP inhibition of ATPase results in improved homeostasis of glycolytic metabolites, ATP and ADP in response to PFK activation, whereas stronger activation of PFK by AMP improves the homeostasis of these three quantities in response to ATPase activation. The results are further evidence of the potential for physiological ATP demand to exert control over glycolytic flux, but additionally show that the known effector interactions, in addition to their previously known role in ATP regulation, could contribute to the remarkable homeostasis of glycolytic-metabolite levels observed in vivo. They further indicate that quantitative characterisation of likely domains of behaviour of metabolic systems can be achieved by an algebraic analysis that is not highly dependent on a full and precise knowledge of the molecular details of the kinetic/regulatory properties of the enzymes, but that still allows an assessment of whether hypotheses regarding the system are feasible and sufficient to account for the observations.

Adenine Nucleotides↗
PubMed1997

Finite change analysis of glycolytic intermediates in tuber tissue of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase.

Genetically engineered organisms overexpressing phosphofructokinase (PFK), a supposed 'regulatory' step of glycolysis, often show little or no measurable change in glycolytic or respiratory flux, although the concentrations of glycolytic intermediates may change. We have used the finite change theory of Metabolic Control Analysis (MCA) to analyse the concentrations of glycolytic metabolites in aged disks of tuber tissue from four lines of transgenic potatoes expressing different amounts of PFK that, under aerobic conditions, showed statistically indistinguishable rates of respiration. The constancy of the metabolites' concentration deviation indices for different increases in PFK expression indicated that the metabolite changes from a graded series, excluding the possibility of anomalous behaviour that might be observed in a single transgenic line. Consequently we were able to use the finite change method to validate the results of an MCA model of tuber glycolysis [Thomas, Mooney, Burrell and Fell (1997) Biochem. J. 322, 119-127]. Furthermore the metabolite changes with PFK activity are evidence that near-equilibrium steps do not transmit increased substrate concentrations down the pathway without attenuation. Our results support the view that flux increase by activation of a single enzyme early in the pathway will, contrary to expectations, be of limited effectiveness in achieving flux increases.

Energy Metabolism↗
PubMed1997

Metabolic Control Analysis of glycolysis in tuber tissue of potato (Solanum tuberosum): explanation for the low control coefficient of phosphofructokinase over respiratory flux.

We have applied Metabolic Control Analysis (MCA) in an attempt to determine the distribution of glycolytic flux control between the steps of glycolysis in aged disks of potato tuber under aerobic conditions, using concentrations of glycolytic metabolites in tuber tissue from a range of transgenic potato plants and published enzyme kinetic data. We modelled the substrate and effector kinetics of potato tuber phosphofructokinase (PFK) by reanalysing published results. Despite the scarcity of reliable kinetic data, our results are in agreement with experimental findings namely that, under the conditions described, PFK has little control over glycolytic flux. Furthermore our analysis predicts that under these conditions far more control lies in the dephosphorylation of phosphoenolpyruvate and/or in the steps beyond. We have validated the results of our analysis in two ways. First, predictions based on calculated concentration control coefficients from the analysis show generally good agreement with observed metabolite deviation indices discussed in the preceding paper [Thomas, Mooney, Burrell, and Fell (1997) Biochem. J. 322, 111-117]. Second, sensitivity analysis of our results shows that the calculated control coefficients are robust to errors in the elasticities used in the analysis, of which relatively few need to be known accurately. Experimental and control analysis results agree with previous predictions of MCA that strong co-operative feedback inhibition of enzymes serves to move flux control downstream of the inhibiting metabolite. We conclude that MCA can successfully model the outcome of experiments in the genetic manipulation of enzyme amounts.

Electron Transport↗
PubMed1996

Design of metabolic control for large flux changes.

Metabolic Control Analysis has invalidated many traditional biochemical concepts of control, in particular the rate-limiting step. However, it has not been used to question the mechanisms by which pathway flux is thought to be controlled, such as the action of allosteric effectors or of covalent modification mechanisms. Here we use Control Analysis and computer simulation to examine the response of pathway segments to change in flux imposed by action on an enzyme outside the segment. Whether these segments contain near-equilibrium enzyme-catalysed reactions, cooperative enzymes, feedforward activation loops or feedback inhibition loops, their responses are significantly different from those observed in vivo. In particular, they do not exhibit the remarkable degrees of metabolite homoeostasis during large flux changes that have frequently been observed experimentally. On the other hand, near-constant levels of metabolites in spite of large changes of flux are consistent with our recent proposal that multi-site modulation--simultaneous activation of many pathway steps-is the normal method by which metabolism is controlled.

Animals↗