Adenylate Charge

 The adenylate charge, or adenylate energy charge, is a linear measure of the energy stored in the adenylate system ([ATP] + [ADP] + [AMP]) in a living cell. It is analogous to the charge of a storage battery. Its value is defined by the expression: 

The status of the adenylate system is best described by the adenylate charge and appears to be the most ubiquitous regulatory signal in metabolism; it affects the rates of nearly all metabolic conversions and the partitioning of metabolically available substances between oxidation, synthesis of cell substance, and production of storage compounds. In living organisms, the same controls that regulate metabolic partitioning also maintain the value of the adenylate charge near 0.9.

 1. Background

Organisms are exquisitely regulated material- and energy-transducing systems. Material from the environment must be converted to the many compounds of which the organism is made, and energy, usually obtained either by absorption of sunlight or from oxidation of foodstuffs, must be converted for use in biosynthesis, movement, and membrane activities. Partitioning of material and energy between those functions must be adjusted continuously to meet the changing needs of the cell or organism. Transduction of energy through the adenylate system is an integral part of each of those functions.

 Any system that is not at chemical and physical equilibrium can, in principle, supply energy. The two types of nonequilibrium situations that are mainly used in energy storage and transduction by organisms are (1) difference in electric charge or chemical potential across a membrane (see Membrane Potentials) and (2) a ratio of [ATP] to [ADP] that is far from equilibrium. All cells contain enzyme systems that catalyze transfer of energy between the two.

The energy status of a chemical storage system may be defined by either of two parameters: the molar Gibbs free energy change of the relevant chemical reaction or the mole fraction of the higher-energy state of the system. The two are computationally interconvertible but are not linearly related. The molar free energy change is a function of the ratio of activities of the products and reactants of the reaction; the mole fraction is a linear measure of the extent of reaction. In the familiar case of a lead storage battery, the free energy is measured by the voltage and the mole fraction by the charge (measured by a hydrometer in this case).

If a generic energy-transducing reaction is indicated by the type of reaction AB, the molar free energy change is given by

and the fractional charge (mole fraction of the more energetic state) by

In the case of a lead storage battery, A is (2Pb(SO₄) + 2H₂O) and B is (Pb + PbO₂ + 2H₂SO₄). For the metabolic adenylate system, A is (ADP + P_i) and B is (ATP).

A system is fully discharged when only A is present and fully charged when it consists of B alone. For intermediate states, the stoichiometrically linear charge function indicates how much work is available as a fraction of that of a totally charged system. [This metabolic use of the term “work” is slightly nonstandard. In thermodynamic terms, the maximum work available is the integrated product of the charge multiplied by the changing value of the Gibbs free energy as the system goes to equilibrium. But the energy of the adenylate system is used stoichiometrically—for example, to affect chemical change or mechanical movement—and charge is therefore proportional to available work, defined in terms of biological effect.] It is because of its stoichiometric nature that the charge function is more relevant in most contexts than the molar free energy change for both storage batteries and the metabolic adenylate system.

If the adenylate system merely alternated between ADP and ATP, the charge function would be the simple ATP mole fraction, [ATP]/([ATP] + [ADP]). Some enzymes, however, couple the use of ATP to synthetic reactions by converting ATP to AMP and pyrophospate, and so the concentration of AMP must also be taken into account. 

The pyrophosphate is rapidly hydrolyzed enzymically by pyrophosphatase, and this removal of product pulls the reaction forward (increases the numerical value of the negative free energy change).

Accumulation of AMP as a consequence of such reactions would deplete the cell's stores of ATP and ADP and so would be rapidly lethal. That outcome is prevented by the action of adenylate kinase, which catalyzes the phosphorylation of AMP: 

The sum of reactions (2) and (3) is conversion of two molecules of ATP to two of ADP. In such cases, a single molecule of ATP, in effect, supplies twice as much metabolic energy to a metabolic reaction as is obtained from action of an ordinary kinase that converts ATP to ADP, but a second molecule of ATP is required to make good the energy balance.

Because of the participation of AMP in metabolic energy transduction, the simple mole fraction of ATP is not an adequate measure of the energy status of the adenylate system. Reaction (3) shows that two molecules of ADP are energetically equivalent to one of ATP. Thus, the linear charge function for the adenylate system (the effective mole fraction of ATP) is seen to be

The adenylate charge is the linear measure of the metabolic work available. A system containing only ATP is fully charged, with an adenylate charge value of 1.0, and one containing only AMP is fully discharged, with an adenylate charge of 0. The charge value would be 0.5 if only ADP were present. If reaction (3) catalyzed by adenylate kinase is at equilibrium, the concentrations of ATP, ADP, and AMP are fixed for any particular value of the adenylate charge (Figure 1).

Figure 1. Mole fractions of the components of the adenylate nucleotide system as a function of the adenylate charge. The reaction catalyzed by adenylate kinase is assumed to be near equilibrium, with an apparent equililbrium constant irrespective of differential ionization and magnesium binding) of 0.8.

2. Regeneration and utilization of ATP

In aerobic organisms, ATP is regenerated mostly by oxidation of foods or storage compounds to carbon dioxide (see ATP Synthase). In anaerobic organisms, substrates undergo other energy-yielding conversions—for example, fermentation of glucose to ethanol and carbon dioxide—rather than oxidation. Carbohydrates are converted to pyruvate by way of the glycolytic pathway. The pyruvate is oxidized to a derivative of acetic acid, acetyl coenzyme A, which is oxidized to carbon dioxide in the reactions of the citrate cycle, or Krebs cycle. The electrons lost in those oxidations are passed on to oxygen by mediation of a series of membrane-associated enzymes. Those electron transfers are coupled to the conversion of ADP to ATP, thus supplying metabolically available energy to the adenylate system. The routes by which other classes of foods are metabolized feed into this central pathway. Fats are broken down to produce acetyl coenzyme A, which joins the carbohydrate pathway at that point. Amino acids derived from protein degradation are metabolized by individual pathways to produce various intermediates of glycolysis or the citrate cycle. Thus, the same central pathways are taken in the utilization of all foods.

Glycolysis and the citrate cycle are also centrally involved in biosynthesis. The biosynthetic pathways leading to the many components of a cell all begin with intermediates of glycolysis or the citrate cycle. That is, 10 or 12 intermediates of these degradative pathways are also the starting points for all synthetic sequences. Each such intermediate occupying a metabolic branchpoint must be partitioned between two competing pathways, one leading to oxidation to carbon dioxide and the other to synthesis of one or more components of the cell. The adenylate system provides energy for the chemical activities of the cell, including biosynthesis, for active transport of nutrients and ions across membranes against chemical potential gradients, and for most other biological requirements, including mechanical movement. All those functions are catalyzed or affected by proteins. Proteins involved in those functions have evolved affinities for ATP and ADP (or AMP) that maximize their functional usefulness to the organism. Thus, it is essential that the ratio of ATP to ADP remain virtually constant. The problem would be equivalent to the regulation of voltage by the power supply of a complex electronic device, if it were not so much more complicated.

3. Regulation of the Regeneration and Utilization of ATP

 The rate of regeneration of ATP from ADP is regulated in large part by controlling the rate at which substrate is made available to the electron transport phosphorylation system. At least five enzymes that catalyze reactions in glycolysis or the citrate cycle respond sensitively to the status of the adenylate pool and adjust the properties of the catalytic site accordingly. An increase in the energy charge causes a decrease in the rate of the reaction catalyzed by the enzyme, and a decrease in charge causes an increase in rate. This feedback inhibition system acts to adjust the rate of regeneration of ATP to meet momentary requirements and thus to stabilize the value of the charge. The multiplicity of control sites may be surprising; a single throttle point would seem to be sufficient to regulate the rate of supply of substrate to the electron transport system. The regulatory requirements are, however, much more complex because the central pathways also supply starting materials for synthesis.

Core metabolism consists of the central pathways by which foodstuffs or storage materials of all types are prepared for oxidation and of branches from those pathways that lead to synthesis of one or more products. The primary function of oxidative pathways is regeneration of ATP from ADP, which puts energy into the adenylate system. Biosynthesis is powered by conversion of ATP to ADP, which removes energy from the adenylate system. Adjustment of the partitioning of resources between these oppositely directed pathways to meet the momentary metabolic needs of the cell or organism is the most central of the regulatory requirements that underlie cellular function and survival. At each branchpoint, the partitioning of resources must respond at least to the energy status of the cell as reflected in the ATP/ADP system and to the momentary need for the end products of the synthetic branch.

At such branchpoints, the next enzyme in the degradative pathway and the first enzyme in the biosynthetic branch compete for the branchpoint metabolite, their common substrate. Maintenance of an appropriate balance between oxidation and biosynthesis requires that both enzymes must be regulated. As well as decreasing the rate of the degradative reaction, an increase in the value of the charge leads to an increase in the rate of the competing reaction that channels the branchpoint metabolite into biosynthesis. Thus, enough substrate is oxidized to maintain the normal status of the adenylate system, and biosynthesis is allowed to the extent that the products are needed and the supply of resources allows.

This partitioning is not affected by turning enzymes on and off but by changing the affinities of the competing enzymes for the substrate, which allows for much more sensitive control. Affinity is usually expressed in terms of the Michaelis constant, K_m, the concentration of substrate at which half the catalytic sites bind substrate, leading to a reaction velocity half the maximal rate. Thus, competition between the enzymes is regulated in the most direct and effective way—by modulating their relative abilities to capture the common substrate. The competition is shown generically in Figure 2. Reactions in pathways that lead to degradation of substrate and the regeneration of ATP respond to variation in the energy status of the adenylate pool as indicated by curve R, and reactions that direct substrate into biosynthetic pathways that utilize ATP respond as shown by curve U. The result is that the value of the energy charge is maintained within a narrow range of values near the intersection of the curves. If the charge drifts upward slightly, the rate of use of ATP in biosynthesis tends to increase and the rate of regeneration of ATP decreases, counteracting the drift. A downward drift has the opposite effect.

Figure 2. Generalized illustration of the effects of the adenylate charge on the rates of reactions in which ATP is regenerated (R) and in which ATP is utilized (U). Curve U was calculated for an enzyme for which the K_m for ATP at the catalytic site is six times that of ADP. Curve U* represents 80% depression of rate as a consequence of feedback inhibition of the enzyme by the end product of the biosynthetic sequence.

The regulatory interactions illustrated by curves R and U are necessary but not adequate. By themselves, they would adjust the rate of all biosynthetic sequences similarly and only on the basis of the availability of energy, without regard to the need for the individual products. Those controls are supplemented by product feedback inhibition. The first enzyme in nearly every biosynthetic sequence that has been studied bears a regulatory site that binds the end product of the sequence. When that site is occupied, the conformation of the enzyme changes so as to decrease the affinity for substrate at the catalytic site (increase the Km) (see Allostery). Thus, as the concentration of end product rises, the fraction of enzyme molecules that bind it increases, the affinity of enzyme for substrate decreases, and the enzyme competes less vigorously for substrate. When the concentration of end product falls—for example, when an amino acid is being used more rapidly for protein synthesis—a smaller fraction of the regulatory sites of the first enzyme involved in its synthesis is occupied by end product. The resulting decrease in Km causes the substrate to be more successful in its competition with the enzyme that catalyzes the next step in the degradative pathway, and the rate of synthesis increases. Such interactions adjust synthetic rates to meet changing metabolic needs. Curve U* in Figure 2 illustrates the response of an enzyme when the end product of its sequence is available in adequate amount from external sources. Any response between curve U* and somewhat above curve U is possible. Under ordinary conditions, the response will fluctuate in the vicinity of curve U to adjust production to meet metabolic demand. These interactions stabilize the pools of amino acids, assuring that they will neither be depleted when demand for protein is high nor build up to unnecessary or injurious levels when demand is small. Pool levels of other metabolites are regulated similarly.

The curves of Figure 2 thus provide a general overview of the regulation of the central pathways in their roles of regenerating ATP and providing starting materials for biosynthesis (1). The interaction of curves R and U adjust degradative metabolism and overall biosynthetic rates so as to maintain a nearly constant value of energy charge and, in the process, necessarily cause an appropriate partitioning of resources between degradation and synthesis. Superposition of product feedback inhibition, illustrated by curve U*, adjusts the rate of each individual synthetic sequence to meet the momentary needs of the organism or cell. Even when the overall rate of biosynthesis is high, the synthetic pathway leading to any given metabolite will be suppressed when that product is in good supply. Thus, the components of this control system act together to assure that the adenylate system, the immediate energy source for nearly all cell activities, is maintained at a high and constant charge, near 0.9 (2), and that the rate of production of each biosynthetic product is determined by interaction between the overall availability of resources and the cell's need for the individual product. Regulation of biosynthetic rates is roughly equivalent to the decisions made by a human consumer who must balance how strongly an item is desired and how readily it can be afforded.

4.Regulatory Properties of Enzymes

Enzymes that show R-type responses (see Allostery) catalyze reactions in the pathways that supply substrates for electron transport–linked regeneration of ATP but usually do not involve ATP or ADP directly. Such enzymes have, therefore, evolved regulatory, allosteric sites, distinct from the catalytic site, where ADP or AMP can bind, causing conformational changes that increase the binding affinity for the substrate at the catalytic site. Some such enzymes decrease substrate affinity when ATP binds at a regulatory site. Typically, the nucleotide binds cooperatively to two or more sites, and the effect on K_m is related to the square or higher power of the nucleotide concentration. Such interactions underlie the sensitive response of R-type enzymes to variation in the value of the charge (3).

 In contrast, ATP and ADP are directly involved in most of the reactions at branchpoints that direct substrate into synthetic sequences. Thus, separate nucleotide-binding regulatory sites are not required. The U-type response is a consequence of higher affinity at the catalytic site for ADP, a product of the reaction, than for ATP, a reactant. This reversal of the usual pattern of higher affinity for reactants than for products results in pronounced inhibition by ADP across most of the energy charge range. At a charge of 0.5, for example, when the concentrations of ATP and ADP are approximately equal, ATP would be excluded from most of the catalytic sites because of competition by ADP. The result would be that most of the kinetic response to variation in the ATP/ADP ratio would occur in a rather narrow range near the high end of the energy charge scale. Curve U of Figure 2 is calculated for an enzyme for which the value of K_m for ATP is six times that for ADP.

The regulatory stability illustrated by Figure 2 depends on (1) precise evolutionary adjustment of the affinities for adenylates at regulatory sites of R-type enzymes and at catalytic sites of U-type enzymes and of the conformational links by which binding at those sites causes conformational changes that modulate affinity for substrate at catalytic sites and (2) the relative affinity at catalytic sites of U-type enzymes for ATP and ADP. As a consequence of such interactions, the ATP/ADP ratio, or the energy charge, affects reaction rates more strongly than do the absolute concentrations of the adenylate nucleotides. A mutant strain of Escherichia coli unable to synthesize adenylate nucleotides grew on adenine-limiting media at essentially normal rates when the intracellular concentrations of ATP, ADP, and AMP were half the normal levels. The energy charge and the ATP/ADP ratio retained their normal values. That mutant, like other organisms that have been studied, did not grow if the energy charge fell slightly below its usual value of about 0.9 (4.(    

The controls illustrated in Figure 2 should not be confused with thermodynamic or mass-action effects; they are strictly kinetic. The value of the Gibbs free energy change is high and negative for U-type reactions under all physiological conditions, as is also true for R-type reactions. Thus, degradation of fuels and synthesis of products are both thermodynamically favorable at all times, and evolved kinetic control mechanisms determine which conversions actually occur. The adenylate energy transduction system, which links metabolic sequences and nearly all other cell activities energetically, is ideally placed for the additional role of mediating the regulatory interactions that underlie the integrated activities of cells and organisms.

References

1. D. E. Atkinson (1972) In Horizons of Biochemistry (A. San Pietro and H. Gest, eds.), Academic Press, New York, pp. 83–96. 

2. A. G. Chapman and D. E. Atkinson (1977) In Advances in Microbiological Physiology, vol. 15 (A. H. Rose and D. W. Tempest, eds.), Academic Press, New York, pp. 253–308. 

3. D. E. Atkinson (1970) In The Enzymes, 3rd ed., vol. 1 (P. D. Boyer, ed.), Academic Press, New York, pp. 461–489. 

4. J. S. Swedes, R. J. Sedo, and D. E. Atkinson (1975) J. Biol. Chem. 250, 6930–6938.