Kinetics of metabolic pathways. A system <i>in vitro</i> to study the control of flux

Torres, Néstor V.; Mateo, Fátima; Meléndez-Hevia, Enrique; Kacser, H.

doi:10.1042/bj2340169

Cited by 99 publications

(55 citation statements)

References 30 publications

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“…Note that, if all enzymes are to be changed by the same factor (i.e. r, = r for all Ej) then Eqn (25) reduces to Eqn (24).…”

Section: Predictions Of Changes In Fluxmentioning

confidence: 99%

“…The case where u, is not proportional to E,, because of enzyme-enzyme interactions for example, can still be treated by an extension of the method we described previously [34, 351. x,+ S I * s, ;-" s, ---*Sn-1- 2 These simplifying assumptions have previously I been found useful [17,18,[25][26][27][28]361 in revealing certain properties of multi-enzyme systems and we shall use them throughout the following treatment where the relationship between the deviation index and the control coefficient is investigated. It should be remembered that the theorems of metabolic control analysis used here do not depend on any linear assumptions.…”

Section: G=(%)mentioning

confidence: 99%

“…1 shows a possible relationship between the flux, J, through a metabolic pathway and the activity of one of the enzymes, E,. In general, that relationship is not known, although in some systems it can be established experimentally [14,[25][26][27][28]. The following terms are defined: E p = original concentration of enzyme E,; r = a factor (non-infinitesimal) by which the enzyme concentration is multiplied to give the 'large' change; r can be greater or less than 1.…”

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Responses of metabolic systems to large changes in enzyme activities and effectors

Small

Kacser

1993

European Journal of Biochemistry

154

124

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This first paper in a series investigates the problem of predicting and analysing the effects of large changes in enzyme activities or external nutrients/effectors on metabolic fluxes. We introduce the concept of a deviation index, D, which gives a measure of the relative change in a metabolic variable (e.g. flux) due to a large (non-infinitesimal) relative change in a parameter (e.g. enzyme). Using simplifying kinetic assumptions we have found, for an unbranched metabolic chain, a direct relationship between deviation indices and flux control coefficients. This relationship provides a method to estimate flux control coefficients using a single large change in enzyme activity. We also provide a method of predicting the effects of, for example, DNA manipulation or other techniques for enzyme activity/concentration changes on metabolic fluxes. Up-modulations of single enzymes rarely produce significant changes in fluxes. We show that combined changes of activity of a group of enzymes will produce a more than 'additive' response. We provide a method of predicting the effects of these combined changes, given either the flux control coefficients of the group of enzymes or the effects on the flux of changing the enzymes individually. A similar analysis is carried out for large changes in external nutrients or effectors. These amplification factors, f, give experimentally accessible estimates of the expected changes in metabolic variables. We provide three 'case studies' to illustrate our results.The investigation of metabolism has had two principal aims : to understand how organisms work and to change them in desired directions. An understanding will be achieved if the physiological responses of the organism can be described in terms of the many thousand of 'unit' parts; the enzymecatalysed reactions. These responses happen as a result of naturally occurring changes in external conditions or due to the experimenter's manipulations. The desire to change organisms, including man, is testified by the long history of medicine and agriculture: to change sickness into health, to change low yields into high yields.The well-known complexity of metabolic structures along with many experimental explorations has made it evident that simple predictions of the outcome of external, or internal, manipulations are rarely possible. This is so in spite of the considerable progress in the enzymology and molecular biology of individual steps in the system. It is now accepted that a solution must come by taking a systemic view, rather than the study of isolated parts.The development of genetic manipulation technology has high-lighted the possibility of altering the metabolic properties of organisms in specific ways. In particular, the possible use of DNA manipulation in order to increase the concenCorrespondence to H. Kacser,

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“…Note that, if all enzymes are to be changed by the same factor (i.e. r, = r for all Ej) then Eqn (25) reduces to Eqn (24).…”

Section: Predictions Of Changes In Fluxmentioning

confidence: 99%

Section: G=(%)mentioning

confidence: 99%

mentioning

confidence: 99%

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Responses of metabolic systems to large changes in enzyme activities and effectors

Small

Kacser

1993

European Journal of Biochemistry

154

124

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“…1, has the operational implication of modulating the concentration of that enzyme. However, direct titration of the amount of an enzyme is possible only in a reconstituted system [50].…”

Section: Experimental Methods For Determining the Control Coefficientsmentioning

confidence: 99%

Control theory of metabolic channelling

1995

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Various factors appear to control muscle energetics, often in conjunction. This calls for a quantitative approach of the type provided by Metabolic Control Analysis for intermediary metabolism and mitochondrial oxidative phosphorylation. To the extent that direct transfer of high energy phosphates and spatial organization plays a role in muscle energetics however, the standard Metabolic Control Theory does not apply, neither do its theorems regarding control. This paper develops the Control Theory that does apply to the muscle system. It shows that direct transfer of high energy phosphates bestows a system with enhanced control: the sum of the control exerted by the participating enzymes on the flux of free energy from the mitochondrial matrix to the actinomyosin may well exceed the 100% mandatory for ideal metabolic pathways. It is also shown how sequestration of high energy phosphates may allow for negative control on pathway flux. The new control theory gives methods functionally to diagnose the extent to which channelling and metabolite sequestration occur. (Mol Cell Biochem 143: 151-168, 1995)

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“…[41][42][43] The dependence of flux on activity or concentration of a single enzyme within a metabolic pathway is identical ͑e.g., an enzyme variant may have half the activity as wild type, or the system has half the enzyme concentration, the resulting effect on flux is the same regardless͒. By examining the extremes of the flux through metabolic networks, it is possible to appreciate the relationship of AK activity to cellular fitness.…”

Section: E the Hill Function Fitness Model Is Consistent With Metabomentioning

confidence: 99%

Evolution of a single gene highlights the complexity underlying molecular descriptions of fitness

Peña

Itallie

Bennett

et al. 2010

Chaos: An Interdisciplinary Journal of Nonlinear Science

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Evolution by natural selection is the driving force behind the endless variation we see in nature, yet our understanding of how changes at the molecular level give rise to different phenotypes and altered fitness at the population level remains inadequate. The reproductive fitness of an organism is the most basic metric that describes the chance that an organism will succeed or fail in its environment and it depends upon a complex network of inter-and intramolecular interactions. A deeper understanding of the quantitative relationships relating molecular evolution to adaptation, and consequently fitness, can guide our understanding of important issues in biomedicine such as drug resistance and the engineering of new organisms with applications to biotechnology. We have developed the "weak link" approach to determine how changes in molecular structure and function can relate to fitness and evolutionary outcomes. By replacing adenylate kinase ͑AK͒, an essential gene, in a thermophile with a homologous AK from a mesophile we have created a maladapted weak link that produces a temperature-sensitive phenotype. The recombinant strain adapts to nonpermissive temperatures through point mutations to the weak link that increase both stability and activity of the enzyme AK at higher temperatures. Here, we propose a fitness function relating enzyme activity to growth rate and use it to create a dynamic model of a population of bacterial cells. Using metabolic control analysis we show that the growth rate exhibits thresholdlike behavior, saturating at high enzyme activity as other reactions in the energy metabolism pathway become rate limiting. The dynamic model accurately recapitulates observed evolutionary outcomes. These findings suggest that in vitro enzyme kinetic data, in combination with metabolic network analysis, can be used to create fitness functions and dynamic models of evolution within simple metabolic systems. © 2010 American Institute of Physics. ͓doi:10.1063/1.3453623͔ Adaptation is the essence of Darwin's brilliant conception of natural selection and is the mechanism by which all organisms are shaped by their environment. Adaptation is a fundamental property of all life and is responsible for recent increases in the populations of drug resistant pathogens that directly impact human health. While microorganisms have relatively simpler and smaller genomes than humans, they still contain thousands of genes that interact with each other to create large networks whose response to challenges is remarkably complex and largely unpredictable. The genome defines the ways in which an organism can respond to its environment and is also a record of adaptation that encodes the "current state" of the organism. The genome provides an excellent system for recording the steps of adaptation at a molecular level (molecular evolution). The challenge of understanding molecular evolution can be addressed through the development of robust experimental systems that afford the opportunity to build and validate models for adaptation....

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Kinetics of metabolic pathways. A system in vitro to study the control of flux

Cited by 99 publications

References 30 publications

Responses of metabolic systems to large changes in enzyme activities and effectors

Responses of metabolic systems to large changes in enzyme activities and effectors

Control theory of metabolic channelling

Evolution of a single gene highlights the complexity underlying molecular descriptions of fitness

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