Kinetics of metabolic pathways. Transient response of the glycolytic system after phosphofructokinase reaction to ADP input

1. Transient response of the glycolytic system, after phosphofructokinase reaction, to ADP input was studied in a metabolic model in vitro, obtained from rat muscle. 2. Under ADP addition to the system, NADH concentration is largely enhanced to be decreased later, showing a very characteristic pulse whose parameters can be used to describe some features of the transition state. 3. NADH pulse was repeatedly obtained by means of successive ADP inputs to the system. 4. The effect of different variables on NADH pulse was studied, these results suggesting a possible role for different steps on this response. 5. The possible relation between the pulse of NADH and oscillatory behaviour of glycolysis is discussed.     

Oscillations and efficiency in glycolysis

We suggest that temporal oscillations of concentrations of intermediates in biochemical reaction systems may enhance the efficiency of free energy conversion (reduce dissipation) in those reactions. Experiments on glycolysis are used to estimate the Gibbs free energy changes along the glycolysis mechanism, and to postulate a construct for the glycolysis "machine" which involves: the PFK reaction as the primary oscillophor; the GAPDH reaction as a phase-shifting device; and the PK reaction with the property of intrinsic oscillatory response at resonance with the driving frequency. Analysis of a simple reaction mechanism with these postulated properties shows that the conversion of free energy from reactants to products is more efficient in an oscillatory than a steady state operation. The efficiency of free energy conversion in glycolysis from glucose + ADP to products + ATP is estimated to be increased by 5--10% due to oscillations. This may have been relevant for the evolutionary development of oscillations such as in glycolysis, especially in anaerobic cells.     

A simulation study of oscillating glycolysis: a comparison between a model and experiments

Glycolysis is the best known biochemical oscillator and suitable for the exploration of the basic features of biological rhythms on a biochemical and mathematical level. Because of our detailed knowledge of its component structure (enzyme kinetics, metabolite pattern) glycolysis can be described by a set of coupled nonlinear differential equations of first order with respect to time, whose individual terms consist of enzyme velocities assuming a steady state hypothesis for the enzymatic forms. Due to the feedback control of PFK by the adenylates the system is able to oscillate. The outcomes of a simplified model, containing only the basic elements of glycolysis, show good agreements with experimental results.     

The response of oscillating glycolysis to perturbations in the NADH/NAD system: a comparison between experiments and a computer model.

The glycolytic pathway is described by a set of coupled non linear differential equations of first order with respect to time. The individual terms of these equations consist of enzyme velocities assuming a steady state hypothesis for the enzymatic forms. These are specified and the system is solved numerically. Oscillations are explained by interaction of PFK with the adenylate system. The conditions for the occurrence of oscillations are tested in a series of computer runs. The phase relations between intermediates of the model agree with those found in yeast cells. As an application of the model the disturbation of oscillations by the addition of acetaldehyde is simulated. The predictions of the model agree with experimental results.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

How to keep glycolytic metabolite concentrations constant when ATP/ADP and NADH/NAD+ change

Glycolytic flux may increase over 100 times in skeletal muscle during rest-to-work transition, whereas glycolytic metabolite concentrations remain relatively constant. This constancy cannot be explained by an identical direct activation of all glycolytic enzymes because the concentrations of ATP, ADP, AMP, P(i), NADH and NAD+, modulators of the activity of different glycolytic enzymes, change. It is demonstrated in the present in silico study that a perfect homeostasis of glycolytic metabolite concentrations can be achieved if glycolysis is divided into appropriate blocks of enzymes that are directly activated to a different extent in order to compensate the effect of the modulators.     

Oscillations of the glycolytic pathway and the purine nucleotide cycle.

In a treatment modeled after the oscillatory behavior of the glycolytic pathway and the purine nucleotide cycle observed in skeletal muscle extracts, it is shown that the basis of the oscillations is the AMP-dependent activation of phosphofructokinase by fructose diphosphate. Control of phosphofructokinase by the adenine nucleotides alone leads to the establishment of a steady state. Whether steady state or oscillatory behavior occurs depends in part on the activity of glyceraldehyde-3-phosphate dehydrogenase, which controls the rate of removal of fructose diphosphate. Under appropriate conditions oscillatory behavior can maintain a higher [ATP]/[ADP] ratio than steady state behavior. Viewed in the context of conditions that may be encountered in skeletal muscle in vivo, oscillatory behavior of glycolysis is shown to have additional advantages for maintaining a high [ATP]/[ADP] ratio.     

Analysis of in vivo kinetics of glycolysis in aerobic Saccharomyces cerevisiae by application of glucose and ethanol pulses

This article presents the dynamic responses of several intra- and extracellular components of an aerobic, glucose-limited chemostat culture of Saccharomyces cerevisiae to glucose and ethanol pulses within a time window of 75 sec. Even though the ethanol pulse cannot perturb the glycolytic pathway directly, a distinct response of the metabolites at the lower part of glycolysis was found. We suggest that this response is an indirect effect, caused by perturbation of the NAD/NADH ratio, which is a direct consequence of the conversion of ethanol into acetaldehyde. This effect of the NAD/NADH ratio on glycolysis might serve as an additional explanation for the observed decrease of 3PG, 2PG, and PEP during a glucose pulse. The responses measured during the ethanol pulse were used to evaluate the allosteric regulation of glycolysis. Our results confirm that FBP stimulates pyruvate kinase and suggest that this effect is pronounced. Furthermore, it appears that PEP does not play an important role in the allosteric regulation of phosphofructo kinase.     

Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation

Activation of glycolysis by insulin in cultured rat hepatocytes is preceded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructose 2,6-bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated. 1. 50 microM ATP completely prevented the 3-5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 microM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin cells with initial high Fru(2,6)P2 content. 2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 microM ATP or 15 microM adenosine 5'[beta, gamma-methylene]triphosphate. ADP and adenosine-5-[gamma-thio]triphosphate were as effective as ATP, whereas 100 microM adenosine 5'[alpha, beta-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis. 3. Extracellular ATP (100 microM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase. 5. 100 microM ATP elevated the cAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level. It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.     

Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei

A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.     

ADP modulates the dynamic behavior of the glycolytic pathway of Escherichia coli

A mathematical model that includes biochemical interactions among the PTS system, phosphofructokinase (PFK), and pyruvate kinase (PK) is used to evaluate the dynamic behavior of the glycolytic pathway of Escherichia coli under steady-state conditions. The influence of ADP, phosphoenolpyruvate (PEP), and fructose-6-phosphate (F6P) on the dynamic regulation of this pathway is also analyzed. The model shows that the dynamic behavior of the system is affected significantly depending on whether ADP, PEP, or F6P is considered constant a steady state. Sustained oscillations are observed only when dADP/dt not equal 0 and completely suppressed if dADP/dt = 0 at any steady-state value. However, when PEP or F6P is constant, the system evolves toward the formation of stable limit cycles with periods ranging from 0.2 min to hours.     

Mechanisms of the regulation of muscle energy metabolism on oxidation of glucose and fatty acids. A mathematical model

A mathematical model was used to study the role of various allosteric regulatory mechanisms in the oxidation of glucose and fatty acids by muscle energy metabolism. A large number of such mechanisms were shown to be involved in simultaneous oxidation of both substrates: glycolysis is regulated by the ATP/ADP ratio at the phosphofructokinase (PFK) step; the control over pyruvate dehydrogenase is exercised by the NADHm/NADm+ and CoAsAc/CoAsH ratios as well as by the level of pyruvate; the Krebs cycle is regulated by oxaloacetate and citrate concentrations in the citrate synthase reaction and by the ATP/ADP and NADHm/NADm+ ratios in the isocitrate dehydrogenase reaction. The inhibition of PFK and pyruvate dehydrogenase by excess of CoAsAcyl as well as the inhibition of PFK by citrate are additional equivalent regulatory mechanisms. When glucose alone is oxidized, the levels of citrate, CoAsAcyl, NADHm and CoAsAc decrease drastically within the whole range of physiological ATPase loads; the only regulating factors that remain efficient are the ATP/ADP ratio in glycolysis, the level of pyruvate at the pyruvate dehydrogenase step, the ATP/ADP ratio and the levels of CoAsAc, oxaloacetate and isocitrate in the Krebs cycle.     

Mathematical model of carbohydrate energy metabolism. Interaction between glycolysis, the Krebs cycle and the H-transporting shuttles at varying ATPase load

A simple mathematical model for carbohydrate energy metabolism based on the stoichiometic structure of glycolysis, the Krebs cycle and oxidative phosphorylation is proposed. The only allosteric regulation involved in the model is phosphofructokinase activation by AMP. Simple as it is, the model can explain the following properties of carbohydrate metabolism: a drastic rise of the rate of glucose consumption during transition to a higher level of ATPase load; stabilization of ATP and an increase of the steady state rates of glycolysis and oxidation of cytoplasmic NADH by the H-transporting shuttles and of pyruvate in the Krebs cycle with increasing rate of the ATPase load; activation of glycolysis and a decrease of the rate of oxidative phosphorylation following an inhibition of the H-transporting shuttles. The mechanisms of the coordinated changes in the steady state rates of glycolysis, the H-transporting shuttles and the Krebs cycle at varying ATPase load in the cell are discussed.     

Analysis of allosteric mechanisms of suppressing parasitic recirculation in the futile cycle fructose-6-P--fructose-1,6-P2

The uncontrollable substrate recirculation in the central futile cycle (FC) in the carbohydrate energy metabolism fructose-6-P (F6P) in equilibrium or formed from fructose-1,6-P2 (FBP), makes it impossible to maintain a stable level of ATP because of its wasteful expenditure in the cycle reactions which are equivalent to the ATPase reaction and also because of the diversion of FBP from glycolytic phosphorylation of ADP. It follows from the analysis of a mathematical model of the carbohydrate energy metabolism that the allosteric inhibition of fructosebisphosphatase (FBPase) by FBP and AMP leads to suppression of the recirculation in the FC and recovery of the ability of glycolysis to stabilize the level of ATP with high accuracy. The allosteric activation of phosphofrucktokinase (PFK) by AMP couples the expenditure of ATP and F6P in the FC with ATP consumption by a load.     

The biochemical properties and phylogenies of phosphofructokinases from extremophiles

The enzyme phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoprotetus tenax) and plant pyrophosphate-dependent phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.     

Compartmentalization of a unique ADP/ATP carrier protein SFEC (Sperm Flagellar Energy Carrier, AAC4) with glycolytic enzymes in the fibrous sheath of the human sperm flagellar principal piece

The longest part of the sperm flagellum, the principal piece, contains the fibrous sheath, a cytoskeletal element unique to spermiogenesis. We performed mass spectrometry proteomics on isolated human fibrous sheaths identifying a unique ADP/ATP carrier protein, SFEC [AAC4], seven glycolytic enzymes previously unreported in the human sperm fibrous sheath, and sorbitol dehydrogenase. SFEC, pyruvate kinase and aldolase were co-localized by immunofluorescence to the principal piece. A homology model constructed for SFEC predicted unique residues at the entrance to the nucleotide binding pocket of SFEC that are absent in other human ADP/ATP carriers, suggesting opportunities for selective drug targeting. This study provides the first evidence of a role for an ADP/ATP carrier family member in glycolysis. The co-localization of SFEC and glycolytic enzymes in the fibrous sheath supports a growing literature that the principal piece of the flagellum is capable of generating and regulating ATP independently from mitochondrial oxidation in the mid-piece. A model is proposed that the fibrous sheath represents a highly ordered complex, analogous to the electron transport chain, in which adjacent enzymes in the glycolytic pathway are assembled to permit efficient flux of energy substrates and products with SFEC serving to mediate energy generating and energy consuming processes in the distal flagellum, possibly as a nucleotide shuttle between flagellar glycolysis, protein phosphorylation and mechanisms of motility.     

Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, while mitochondrial membrane potential was measured using the fluorescent dye DiOC 2(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP/ADP antiporter inhibitor atractyloside and high concentrations of the ATPase inhibitor N, N'-dicyclohexylcarbodiimide, suggesting that there is a strong coupling between oscillations in mitochondrial membrane potential and oscillations in NADH mediated by the ATP/ADP antiporter and possibly also other respiratory components.     

Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR

The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using (13)C and (31)P NMR to monitor in vivo the kinetics of the pools of NAD(+), NADH, ATP, inorganic phosphate (P(i)), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of (13)C-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH(-) deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (approximately 5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH(-) strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH(-) strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P(i) content could play an important role.