scholarly journals Quantitative determinants of aerobic glycolysis identify flux through the enzyme GAPDH as a limiting step

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Alexander A Shestov ◽  
Xiaojing Liu ◽  
Zheng Ser ◽  
Ahmad A Cluntun ◽  
Yin P Hung ◽  
...  
Keyword(s):  
Aerobic Glycolysis ◽  
Metabolic Control Analysis ◽  
Integrated Analysis ◽  
Control Analysis ◽  
Metabolomics Data ◽  
Limiting Step ◽  
Quantitative Changes ◽  
Fructose 1,6 Bisphosphate ◽  
And Control ◽  
Rate Limiting

Aerobic glycolysis or the Warburg Effect (WE) is characterized by the increased metabolism of glucose to lactate. It remains unknown what quantitative changes to the activity of metabolism are necessary and sufficient for this phenotype. We developed a computational model of glycolysis and an integrated analysis using metabolic control analysis (MCA), metabolomics data, and statistical simulations. We identified and confirmed a novel mode of regulation specific to aerobic glycolysis where flux through GAPDH, the enzyme separating lower and upper glycolysis, is the rate-limiting step in the pathway and the levels of fructose (1,6) bisphosphate (FBP), are predictive of the rate and control points in glycolysis. Strikingly, negative flux control was found and confirmed for several steps thought to be rate-limiting in glycolysis. Together, these findings enumerate the biochemical determinants of the WE and suggest strategies for identifying the contexts in which agents that target glycolysis might be most effective.

scholarly journals Metabolic Control Analysis: A Tool for Designing Strategies to Manipulate Metabolic Pathways

2008 ◽  
Vol 2008 ◽  
pp. 1-30 ◽  
Author(s):  
Rafael Moreno-Sánchez ◽  
Emma Saavedra ◽  
Sara Rodríguez-Enríquez ◽  
Viridiana Olín-Sandoval
Keyword(s):  
Metabolic Control ◽  
Metabolic Pathway ◽  
Large Scale ◽  
Metabolic Control Analysis ◽  
Metabolite Concentration ◽  
Control Analysis ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Experimental Approaches ◽  
Rate Limiting

The traditional experimental approaches used for changing the flux or the concentration of a particular metabolite of a metabolic pathway have been mostly based on the inhibition or over-expression of the presumed rate-limiting step. However, the attempts to manipulate a metabolic pathway by following such approach have proved to be unsuccessful. Metabolic Control Analysis (MCA) establishes how to determine, quantitatively, the degree of control that a given enzyme exerts on flux and on the concentration of metabolites, thus substituting the intuitive, qualitative concept of rate limiting step. Moreover, MCA helps to understand (i) the underlying mechanisms by which a given enzyme exerts high or low control and (ii) why the control of the pathway is shared by several pathway enzymes and transporters. By applying MCA it is possible to identify the steps that should be modified to achieve a successful alteration of flux or metabolite concentration in pathways of biotechnological (e.g., large scale metabolite production) or clinical relevance (e.g., drug therapy). The different MCA experimental approaches developed for the determination of the flux-control distribution in several pathways are described. Full understanding of the pathway properties when working under a variety of conditions can help to attain a successful manipulation of flux and metabolite concentration.

scholarly journals Rate-limiting step and control of coenzyme A synthesis in cardiac muscle.

1982 ◽  
Vol 257 (18) ◽  
pp. 10967-10972 ◽  
Author(s):  
J D Robishaw ◽  
D Berkich ◽  
J R Neely
Keyword(s):  
Cardiac Muscle ◽  
Coenzyme A ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
And Control ◽  
Rate Limiting

scholarly journals Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations1: computer simulation and Metabolic Control Analysis

1999 ◽  
Vol 342 (3) ◽  
pp. 597-604 ◽  
Author(s):  
Peter J. MULQUINEY ◽  
Philip W. KUCHEL
Keyword(s):  
Computer Simulation ◽  
Metabolic Control ◽  
Energy Demand ◽  
Feedback Inhibition ◽  
Oxygen Affinity ◽  
Metabolic Control Analysis ◽  
Product Inhibition ◽  
Control Analysis ◽  
And Control

This is the third of three papers [see also Mulquiney, Bubb and Kuchel (1999) Biochem. J. 342, 565-578; Mulquiney and Kuchel (1999) Biochem. J. 342, 579-594] for which the general goal was to explain the regulation and control of 2,3-bisphosphoglycerate (2,3-BPG) metabolism in human erythrocytes. 2,3-BPG is a major modulator of haemoglobin oxygen affinity and hence is vital in blood oxygen transport. A detailed mathematical model of erythrocyte metabolism was presented in the first two papers. The model was refined through an iterative loop of experiment and simulation and it was used to predict outcomes that are consistent with the metabolic behaviour of the erythrocyte under a wide variety of experimental and physiological conditions. For the present paper, the model was examined using computer simulation and Metabolic Control Analysis. The analysis yielded several new insights into the regulation and control of 2,3-BPG metabolism. Specifically it was found that: (1) the feedback inhibition of hexokinase and phosphofructokinase by 2,3-BPG are equally as important as the product inhibition of 2,3-BPG synthase in controlling the normal in vivo steady-state concentration of 2,3-BPG; (2) H+ and oxygen are effective regulators of 2,3-BPG concentration and that increases in 2,3-BPG concentrations are achieved with only small changes in glycolytic rate; (3) these two effectors exert most of their influence through hexokinase and phosphofructokinase; (4) flux through the 2,3-BPG shunt changes in absolute terms in response to different energy demands placed on the cell. This response of the 2,3-BPG shunt contributes an [ATP]-stabilizing effect. A ‘cost’ of this is that 2,3-BPG concentrations are very sensitive to the energy demand of the cell and; (5) the flux through the 2,3-BPG shunt does not change in response to different non-glycolytic demands for NADH.

scholarly journals Control analysis applied to single enzymes: can an isolated enzyme have a unique rate-limiting step?

1993 ◽  
Vol 294 (1) ◽  
pp. 87-94 ◽  
Author(s):  
G C Brown ◽  
C E Cooper
Keyword(s):  
Rate Constants ◽  
General Assumption ◽  
Control Analysis ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Rate ◽  
Carbamate Kinase ◽  
State Rate ◽  
Rate Limiting ◽  
Control Coefficients

Control analysis is used to analyse and quantify the concept of a rate-limiting step within an enzyme. The extent to which each rate constant within the enzyme limits the steady-state rate of the enzyme and the levels of enzyme intermediate species are quantified as flux and concentration control coefficients. These coefficients are additive and obey summation theorems. The control coefficients of triose phosphate isomerase, carbamate kinase and lactate dehydrogenase are calculated from literature values of the rate constants. It is shown that, contrary to previous assumption, these enzymes do not have a unique rate-limiting step, but rather flux control is shared by several rate constants and varies with substrate, product and effector concentrations, and with the direction of the reaction. Thus the general assumption that an enzyme will have a unique rate-limiting step is unjustified.

Use of metabolic control analysis in lactation biology

2008 ◽  
Vol 146 (3) ◽  
pp. 267-273 ◽  
Author(s):  
T. C. WRIGHT ◽  
J. P. CANT ◽  
B. W. MCBRIDE
Keyword(s):  
Sensitivity Analysis ◽  
Fatty Acid ◽  
Metabolic Control ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Metabolic Control Analysis ◽  
Control Analysis ◽  
Acetyl Coa Carboxylase ◽  
Rate Limiting ◽  
Control Coefficients

SUMMARYSensitivity analysis is routinely carried out in the evaluation of simulation models to identify the degree to which parameters influence model outputs. This type of sensitivity analysis is much less frequently applied to real systems, but a technique called metabolic control analysis (MCA) was developed in the 1970s for the purpose of experimentally identifying the degree to which individual enzymes in a metabolic pathway influence flux through the pathway. MCA is applied to the results of inhibition, activation or genetic manipulation of enzymatic steps in a biochemical pathway. Flux control coefficients for each enzyme are defined as the fractional change in steady-state flux through the entire pathway for an infinitesimal change in the activity of that one enzyme. The sum of control coefficients in a linear, non-branching pathway is equal to one. It is a common finding in MCA that the control, or sensitivity, is distributed over multiple enzymes and not in a single rate-limiting enzyme. The fundamental principles of MCA are reviewed and an overview of experimental methods to measure control coefficients is provided, with the objective of introducing this approach to the fields of agricultural biochemistry and modelling, where it is little known. The application of MCA to the study of glucose metabolism and fatty acid synthesis in bovine mammary tissue are reviewed. The analyses indicated that mammary hexokinase activity exerts more control than transmembrane transport of glucose over lactose synthesis, and that control of cytosolic fatty acid synthesis is shared between acetyl-CoA carboxylase and fatty acid synthase, contrary to the widely held view that acetyl CoA carboxylase is the rate-limiting enzyme. It is suggested that MCA could be a valuable aid in the integration of proteomic and metabolomic data with metabolic flux measurements to engineer desired changes in the composition of milk from dairy animals.

Regulation analysis of energy metabolism.

1997 ◽  
Vol 200 (2) ◽  
pp. 193-202 ◽  
Author(s):  
M D Brand
Keyword(s):  
Steady State ◽  
Energy Metabolism ◽  
Metabolic Control Analysis ◽  
Steady States ◽  
Control Analysis ◽  
Top Down ◽  
Down Regulation ◽  
Metabolic Systems ◽  
And Control ◽  
Control Coefficients

This paper reviews top-down regulation analysis, a part of metabolic control analysis, and shows how it can be used to analyse steady states, regulation and homeostasis in complex systems such as energy metabolism in mitochondria, cells and tissues. A steady state is maintained by the variables in a system; regulation is the way the steady state is changed by external effectors. We can exploit the properties of the steady state to measure the kinetic responses (elasticities) of reactions to the concentrations of intermediates and effectors. We can reduce the complexity of the system under investigation by grouping reactions into large blocks connected by a small number of explicit intermediates-this is the top-down approach to control analysis. Simple titrations then yield all the values of elasticities and control coefficients within the system. We can use these values to quantify the relative strengths of different internal pathways that act to keep an intermediate or a rate constant in the steady state. We can also use them to quantify the relative strengths of different primary actions of an external effector and the different internal pathways that transmit its effects through the system, to describe regulation and homeostasis. This top-down regulation analysis has been used to analyse steady states of energy metabolism in mitochondria, cells and tissues, and to analyse regulation of energy metabolism by cadmium, an external effector, in mitochondria. The combination of relatively simple experiments and new theoretical structures for presenting and interpreting the results means that top-down regulation analysis provides a novel and effective way to analyse steady states, regulation and homeostasis in intricate metabolic systems.

scholarly journals Organization and control of the ascorbate biosynthesis pathway in plants

2020 ◽  
Author(s):  
Mario Fenech ◽  
Vítor Amorim-Silva ◽  
Alicia Esteban del Valle ◽  
Dominique Arnaud ◽  
Araceli G. Castillo ◽  
...  
Keyword(s):  
Interaction Analysis ◽  
Control Point ◽  
Metabolic Control Analysis ◽  
Control Analysis ◽  
Photosynthetic Organisms ◽  
Ascorbate Concentration ◽  
Transgenic Lines ◽  
Mutant Complementation ◽  
And Control ◽  
Ascorbate Biosynthesis

ABSTRACTThe enzymatic steps involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and l-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and l-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and l-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

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