Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

• Published in: Molecular systems biology Vol. 5; no. 1; pp. 271 - n/a
• Main Authors: Bastien, Olivier, Cornish-Bowden, Athel, Dumas, Renaud, Cárdenas, María Luz, Robert-Genthon, Mylène, Curien, Gilles
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 2009; EMBO Press; Nature Publishing Group
• Subjects: Allosteric properties; Allosteric Regulation; Amino acids; Amino Acids - metabolism; Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis - metabolism; aspartate metabolism; Aspartic Acid - metabolism; Biochemistry, Molecular Biology; Chloroplasts; Chloroplasts - chemistry; Chloroplasts - metabolism; Computer Simulation; Dehydrogenases; Enzymes; Fluxes; Isoforms; Kinases; Kinetics; Life Sciences; mathematical model; Mathematical models; Metabolism; Metabolites; Models, Biological; Physiology; Protein synthesis; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Regulation; Reproducibility of Results; simulation; Threonine; Validity; Arabidopsis thaliana; aminoacid; metabolic pathway; flux distribution; threonine; simulation; plant; mathematical model; metabolism; allosteric regulation; aspartate; enzyme kinetics
• ISSN: 1744-4292; 1744-4292
• DOI: 10.1038/msb.2009.29

The aspartate‐derived amino‐acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non‐intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration. Synopsis A challenging goal of systems biology is to develop detailed kinetic models for simulating and predicting the dynamic responses of metabolic networks. However, very few published models have been based on kinetic measurements on enzymes in conditions relevant to those in vivo. In addition, the few kinetic models of real systems that have been published do not address the action of effectors in branched pathways. As feedback regulation often nullifies the effects of genetic manipulations, it is important to understand the molecular mechanism of robustness to genetic perturbations of biochemical networks. Until now the functioning of allosteric controls in a branched metabolic system has only been studied theoretically (e.g. Savageau, 1974; Cornish‐Bowden et al, 1995). In addition, branch‐point enzymes often exist as isoforms that respond unequally to cooperative inhibition (or sometimes activation) by allosteric effectors. The physiological role of isoforms has been discussed for many years (Ureta, 1978), and study of the flux distribution between isoforms in a real pathway should shed light on the need for them. The aspartate‐derived amino‐acid pathway from plants constitutes an excellent model system for understanding regulatory mechanisms in branched metabolic pathways. This pathway is responsible for the distribution of the carbon flux from aspartate into the branches for synthesis of lysine, threonine, methionine and isoleucine. There are several branch‐points, many enzyme isoforms and different allosteric control mechanisms (inhibition, activation, antagonism and synergism), as illustrated in Figure 1B. Much is known about the individual components of the systems but little about their role in the economy of the ensemble, as until now no large‐scale detailed kinetic model of this system has been available either in plants or in microorganisms, though small parts of it have been modelled in bacteria (Chassagnole et al, 2001; Yang et al, 2005) and in plants (Curien et al, 2003). A mathematical model of the core of the pathway in the chloroplasts of the model plant Arabidopsis has been constructed with a view to understanding and quantifying the short‐term regulatory capabilities of the system. Flux values and metabolite concentration calculated with the model (Figure 3) were very close to in vivo values. In addition, the metabolic pattern of several mutants could be reproduced. The present work represents the first detailed kinetic model of a real branched metabolic pathway in which all the allosteric controls and isoforms are taken into account. Kinetic characterization in conditions that mimic the in vivo metabolic context, together with the use of purified recombinant enzymes, explains the predictive power of the model. A key result from this model is the identification of allosteric interactions whose function is not to couple demand and but to maintain a high independence between fluxes in competing pathways. Specifically, it shows that proximal controls of dihydrodipicolinate synthase (DHDPS) by Lys and of threonine synthase (TS1) by S‐adenosylmethionine (AdoMet) are involved in the coupling between demand and supply. Distal controls (synergistic inhibition of aspartate kinase 1, inhibition of aspartate kinase 2 by lysine) may also contribute to this function but are much less efficient. Their main function is rather to attenuate flux changes in the branches where demand remains constant, and they thus participate in the independence between pathways. The existence of isoforms with different regulatory patterns also contributes to this independence indicating that enzyme isoforms are not functionally redundant. Another result is the explanation of threonine concentration instability, suggesting a regulatory role for threonine at a higher level of integration. Finally, important information useful for later work is the estimation of the half‐time of the system (400 s). This metabolic system is a slow responsive one, but kinetic controls are still slightly faster than protein concentration control mechanisms. A key result from the quantitative model of the aspartate pathway from Arabidopsis is the identification of feedback controls whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. The model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and/or to its regulation Another result is the identification of threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration