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Biomedical subjects

B N Kholodenko

Publications and source records attributed to B N Kholodenko.

At least 19 recordsLinked to original sources
PubMed1999

Quantification of short term signaling by the epidermal growth factor receptor.

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.

Animals↗
PubMed1999

Spatial gradients of cellular phospho-proteins.

If a protein is rapidly phosphorylated and dephosphorylated at separate cellular locations and protein diffusion is slow, then a spatial gradient of the phosphorylated form of the protein may develop within the cell. We have estimated the potential size of such gradients using measured values of protein diffusion coefficients and protein kinase and phosphatase activities. We analysed two different cellular geometries: (1) where the kinases is located on the plasma membrane of a spherical cell and the phospatase is distributed homogenously in the cytoplasm and (2) where the kinase is located on one planar membrane and the phosphatase on a second parallel planar membrane. The estimated gradients of phospho-proteins were potentially very large, which has important implications for cellular signalling.

Biological Transport↗
PubMed1998

Implications of macromolecular crowding for signal transduction and metabolite channeling.

The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux-response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of approximately 1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux-response coefficient, in one case to values of approximately 1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux-response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.

Cell-Free System↗
PubMed1998

Metabolic design: how to engineer a living cell to desired metabolite concentrations and fluxes.

A biotechnological aim of genetic engineering is to increase the intracellular concentration or secretion of valuable compounds, while making the other concentrations and fluxes optimal for viability and productivity. Efforts to accomplish this based on over-expression of the enzyme, catalyzing the so-called "rate-limiting step," have not been successful. Here we develop a method to determine the enzyme concentrations that are required to achieve such an aim. This method is called Metabolic Design Analysis and is based on the perturbation method and the modular ("top-down") approach-formalisms that were first developed for the analysis of biochemical regulation such as, Metabolic Control Analysis. Contrary to earlier methods, the desired alterations of cellular metabolism need not be small or confined to a single metabolite or flux. The limits to the alterations of fluxes and metabolite concentrations are identified. To employ Metabolic Design Analysis, only limited kinetic information concerning the pathway enzymes is needed.

Biotechnology↗
PubMed1998

Control analysis of metabolic systems involving quasi-equilibrium reactions.

Reactions for which the rates are extremely sensitive to changes in the concentrations of variable metabolite concentrations contribute little to the control of biochemical reaction networks. Yet they do interfere with the calculation of the system's behaviour, both in terms of numerical integration of the rate equations and in terms of the analysis of metabolic control. We here present a way to solve this problem systematically for systems with time hierarchies. We identify the fast reactions and fast metabolites, group them apart from the other ("slow") reactions and metabolites, and then apply the appropriate quasi-equilibrium condition for the fast subsystem. This then makes it possible to eliminate the fast reactions and their elasticity coefficients from the calculations, allowing the calculation of the control coefficients of the slow reactions in terms of the elasticity coefficients of the slow reactions. As expected, the elasticity coefficients of the fast reactions drop out of the calculations, and they are irrelevant for control at the time resolution of the steady state of the slow reactions. The analysis, when applied iteratively, is expected to be particularly valuable for the control analysis of living cells, where a time hierarchy exists, the fastest being at the level of enzyme kinetics and the slowest at gene expression.

Cells↗
PubMed1998

A model of O2.-generation in the complex III of the electron transport chain.

Oxidation of semiquinone by O2 in the Q cycle is known to be one of the sources of superoxide anion (O2.-) in aerobic cells. In this paper, such a phenomenon was analyzed using the chemical kinetics model of electron transfer from succinate to cytochrome c, including coenzyme Q, the complex III non-heme iron protein FeSIII and cytochromes bl, bh and cl. Electron transfers from QH2 to FeSIII and cytochrome bl were assumed to occur according to direct transfer mechanism (dynamic channelling) involving the formation of FeS(red)III-Q.- and Q.--cytochrome bl complexes. For oxidation/reduction reactions involving cytochromes bh and bl, the dependence of the equilibrium and elementary rate constants on the membrane potential (deltapsi) was taken into consideration. The rate of O2.- generation was found to increase dramatically with increase in deltapsi above the values found in State 3. On the other hand, the rate of cytochrome c reduction decreased sharply at the same values of the membrane potential. This explains experimental data that the O2.- generation at State 4 appears to be very much faster than at State 3. A mild uncoupling in State 4 can markedly decrease the superoxide generation due to a decrease in deltapsi below the above mentioned critical level. DeltapH appears to be equally effective as deltapsi in stimulation of superoxide production which depends, in fact, upon the deltamuH+ level.

Benzoquinones↗
PubMed1998

Subtleties in control by metabolic channelling and enzyme organization.

Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.

Energy Metabolism↗
PubMed1997

Quantification of information transfer via cellular signal transduction pathways.

A conceptual framework is developed for the quantitative analysis of signal transfer through cellular signal transduction pathways and networks. This approach is referred to as signal transfer analysis and is based on formalisms that were first developed for the analysis of metabolic networks. Signal transduction is quantified as the sensitivity, known as the response coefficient of a target (e.g. an ion channel or transcription factor) to a signal (e.g. a hormone, growth factor or neurotransmitter). This response coefficient is defined in terms of the fractional change in the activated target brought about by a small fractional change in the signal. Quantifying the signal transduction in this way makes it possible to prove that for an idealized signaling cascade without feedback loops, the total response equals the product of all the local response coefficients, one for each level of the cascade. We show under which conditions merely having more levels in a cascade can boost the sensitivity of a target to a signal. If a signal propagates to a target through two different routes, these routes contribute independently to the total response, provided there is no feedback from the target. This independence makes the behavior of signaling cascades different from that of metabolic pathways, where different branches are connected through Kirchhoffs law. The relations between the total response and the local kinetics at each level are given for a number of network structures, such as branched signaling pathways and pathways with feedback. The formalism introduced here may provide a general approach to quantify cellular information transfer.

Cell Physiological Phenomena↗
PubMed1996

Control analysis of glycolytic oscillations.

The principles involved in the control of the frequency of sustained metabolic oscillations are developed in the context of glycolytic oscillations in Saccharomyces cerevisiae. To this purpose, an existing mathematical model that describes the experimentally obtained oscillations was simplified to a core model. Frequency, relative phase, average concentrations and amplitudes of the oscillations were well approximated by writing the two remaining metabolic variables of the core model (representing [ATP] and [hexose]) as harmonic functions of time and by requiring them to fulfill the differential equations. The extent to which an enzyme (-conglomerate) controls the frequency in a sustained oscillation is defined as the log-log derivative of that frequency with respect to enzyme activity. In both the full model and the core model this control of frequency and the control over the average concentrations proved to be distributed over the enzymes. We identified a summation theorem, stating that the sum of such control coefficients over all processes equals unity for frequency and zero for the average concentrations.

Catalysis↗
PubMed1996

Molecular control analysis: control within proteins and molecular processes.

Molecular control analysis is a method for analysing the extent to which the different elementary steps or rate constants within a molecular process limit the steady-state rate (or other variables) of that process. Any process which may be described by a kinetic diagram of transitions between states of the system may be analysed by molecular control analysis, and this approach has previously been used to analyse control within enzymes, transporters, enzyme complexes, channelled pathways, and group-transfer pathways. We outline the theory of molecular control analysis here, and illustrate its use by analysing control within enzymes (three beta-lactamases). Further potential applications include signal-transduction processes, protein folding, and chemical reactions.

Animals↗
PubMed1996

Strong control on the transit time in metabolic channelling.

A suite of different characteristic times is used to describe the temporal behavior of a metabolic pathway. Here we focus on the 'transit' time, that is the average time it takes for a molecule, entering the steady-state pathway as a substrate, to exit the pathway as a product. We show that metabolic channelling results in dramatic changes in control exerted by pathway enzymes on the transit time. In an 'ideal' pathway a doubling of the enzyme concentrations halves the transit time. In a dynamic channel such an increase can reduce the transit time by a factor of four or more.

Enzymes↗
PubMed1996

Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?

It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties.

Allosteric Regulation↗
PubMed1996

Effect of channelling on the concentration of bulk-phase intermediates as cytosolic proteins become more concentrated.

This paper shows that metabolic channelling can provide a mechanism for decreasing the concentration of metabolites in the cytoplasm when cytosolic proteins become more concentrated. A dynamic complex catalysing the direct transfer of an intermediate is compared with the analogous pathway lacking a channel (an "ideal" pathway). In an ideal pathway a proportional increase in protein content does not result in a change in the steady-state concentration of the bulk-phase intermediate, whereas in a channelling pathway the bulk-phase intermediate either decreases or increases depending on the elemental rate constants within the enzyme mechanisms. When the concentration of the enzymes are equal, the pool size decreases with increasing protein concentration if the elemental step depleting the bulk-phase intermediate exerts more control on its concentration than the step supplying the intermediate. Results are illustrated numerically, and a simplified dynamic channel is analysed in which the concentration of the enzyme-enzyme forms. For such a "hit-and-run" channel it is shown that, when the product-releasing step of the enzyme located upstream is close to equilibrium, the pool size decreases as the concentrations of the enzymes increase in proportion, regardless of the rate, equilibrium constants and concentration ratios of the two sequential enzymes.

Cytosol↗
PubMed1996

[Control of the rate of the glucokinase reaction by its elementary stages].

Limitation of an enzymatic reaction by its elemental steps is characterized by so-called control coefficients for (quasi)stationary reaction rate. Established paradigm of "limiting step" cannot describe the control of glucokinase reaction. Flux control is distributed between several elemental steps (ATP binding, ADP release, etc.); this distribution significantly changes when reaction conditions are modified, i.e., changes in glucose or ATP concentrations occur. At about 1 mM ATP and 2-5 mM glucose, impact of ATP binding stage on total flux control is positive and it exceeds 50% if ATP binds after glucose. However, if ATP binds before glucose then control coefficient for ATP binding is negative. At high substrate concentrations, impact of product dissociation on flux control becomes significant. Distribution of control between elemental steps cannot be predicted by changes of Gibbs energy or by the rate constants of elemental steps.

Adenosine Triphosphate↗
PubMed1995

Calcium indirectly increases the control exerted by the adenine nucleotide translocator over 2-oxoglutarate oxidation in rat heart mitochondria.

The effect of calcium on the control exerted by the adenine nucleotide translocator over respiration in isolated heart mitochondria was investigated in order to determine whether calcium directly stimulates the translocator. At respiration rates intermediate between states 3 and 4, Ca2+ is shown to increase the control over 2-oxoglutarate oxidation exerted by the adenine nucleotide translocator in rat heart mitochondria. This did not occur when succinate was the respiratory substrate, even though the control exerted by the translocator was substantial, indicating that Ca2+ does not have a direct effect on the adenine nucleotide translocator. Ca2+ increased the uncoupled oxidation rate of 2-oxoglutarate, but not succinate. Using the summation theorem for flux control, the effect of Ca2+ is explained by a shift of the control over respiration rate toward the adenine nucleotide translocator, from the respiratory chain, presumably as the result of the activation of the 2-oxoglutarate dehydrogenase complex.

Adenosine Triphosphate↗