Chapter 28

Metabolic Integration and
the Unidirectionality
of Pathways

Ballet class,University of San Francisco.
Metabolic integration is achieved through the
highly regulated choreography of thousands of
enzymatic reactions. (Phil Schermeister/Tony Stone Images)

In the preceding chapters in this section (Part III: Metabolism and Its Regulation), we have explored the major metabolic pathways—glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, photosynthesis, gluconeogenesis, fatty acid oxidation, lipid biosynthesis, amino acid metabolism, and nucleotide metabolism. Several of these pathways are catabolic and serve to generate chemical energy useful to the cell; others are anabolic and use this energy to drive the synthesis of essential biomolecules. Despite their opposing purposes, these reactions typically occur at the same time, so that food molecules are broken down to provide the building blocks and energy for ongoing biosynthesis. Cells maintain a dynamic steady state through processes that involve considerable metabolic flux. We can gain a broader understanding of metabolism and biological processes in general if we step back and consider intermediary metabolism at a systems level of organization. Our goal is to integrate metabolic pathways into a regulated, orderly, responsive whole that is in accord with the vitality and stability of cells.¹

28.1 · A Systems Analysis of Metabolism

The metabolism of a typical heterotrophic cell can be portrayed by a schematic diagram consisting of just three interconnected functional blocks: (1) catabolism, (2) anabolism, and (3) macromolecular synthesis and growth (Figure 28.1).

1. CATABOLISM.  Foods are oxidized to CO₂ and H₂O in catabolism, and most of the electrons liberated are passed to oxygen via an electron transport pathway coupled to oxidative phosphorylation, so that ATP is formed. Some electrons go to reduce NADP⁺ to NADPH, the source of reducing power for anabolism. Glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation, and the pentose phosphate pathway are the principal pathways within this block. The metabolic intermediates in these pathways also serve as substrates for processes within the anabolic block.

2. ANABOLISM.  The biosynthetic reactions that form the great variety of cellular molecules are included in anabolism. For thermodynamic reasons, the chemistry of anabolism is more complex than that of catabolism (i.e., it takes more energy [and often more steps] to synthesize a molecule than can be produced from its degradation). Metabolic intermediates derived from glycolysis and the citric acid cycle are the precursors for this synthesis, with NADPH supplying the reducing power and ATP the coupling energy.

3. MACROMOLECULAR SYNTHESIS GROWTH.  The organic molecules produced in anabolism are the building blocks for creation of macromolecules. Like anabolism, macromolecular synthesis is driven by the energy derived from ATP, although indirectly in some cases: GTP is the principal energy source for protein synthesis, CTP for phospholipid synthesis, and UTP for polysaccharide synthesis. However, keep in mind that ATP is the ultimate phosphorylating agent for formation of GTP, CTP, and UTP from GDP, CDP, and UDP, respectively. Macromolecules are the agents of biological function and information —proteins, nucleic acids, lipids that self-assemble into membranes, and so on. Growth can be represented as cellular accumulation of macromolecules and the partitioning of these materials of function and information into daughter cells in the process of cell division.

Only a Few Intermediates Interconnect the Major Metabolic Systems

Despite the complexity of processes taking place within each block, the connections between blocks involve only a limited number of substances. Just 10 or so kinds of catabolic intermediates from glycolysis, the pentose phosphate pathway, and the citric acid cycle serve as the raw material for most of anabolism: four kinds of sugar phosphates (triose-P, tetrose-P, pentose-P, hexose-P), three a-keto acids (pyruvate, oxaloacetate, and a-ketoglutarate), two coenzyme A derivatives (acetyl-CoA and succinyl-CoA), and PEP (phosphoenolpyruvate).

ATP and NADPH Couple Anabolism and Catabolism

Metabolic intermediates are consumed by anabolic reactions and must be continuously replaced by catabolic processes. In contrast, the energy-rich compounds ATP and NADPH are recycled rather than replaced. When these substances are used in biosynthesis, the products are ADP and NADP⁺, and ATP and NADPH are regenerated from them by oxidative reactions that occur in catabolism. ATP and NADPH are unique in that they are the only compounds whose purpose is to couple the energy-yielding processes of catabolism to the energy-consuming reactions of anabolism. Certainly, other coupling agents serve essential roles in metabolism. For example, NADH and [FADH₂] participate in the transfer of electrons from substrates to O₂ during oxidative phosphorylation. However, these reactions are solely catabolic, and the functions of NADH and [FADH₂] are fulfilled within the block called catabolism.

Figure 28.1 · Block diagram of intermediary metabolism.

Phototrophs Have an Additional Metabolic System—The Photochemical Apparatus

The systems in Figure 28.1 reviewed thus far are representative only of metabolism as it exists in aerobic heterotrophs. The photosynthetic production of ATP and NADPH in photoautotrophic organisms entails a fourth block, the photochemical system (Figure 28.1). This block consumes H₂O and releases O₂. When this fourth block operates, energy production within the catabolic block can be largely eliminated. Yet another block, one to account for the fixation of carbon dioxide into carbohydrates, is also required for photoautotrophs. The inputs to this fifth block are the products of the photochemical system—ATP and NADPH—and CO₂ derived from the environment. The carbohydrate products of this block may enter catabolism, but not primarily for energy production. In photoautotrophs, carbohydrates are fed into catabolism to generate the metabolic intermediates needed to supply the block of anabolism. Although these diagrams are oversimplifications of the total metabolic processes in heterotrophic or phototrophic cells, they are useful illustrations of functional relationships between the major metabolic subdivisions. This general pattern provides an overall perspective on metabolism, making its purpose easier to understand.

28.2 · Metabolic Stoichiometry and ATP Coupling

Virtually every metabolic pathway either consumes or produces ATP. The amount of ATP involved—that is, the stoichiometry of ATP synthesis or hydroly-sis—lies at the heart of metabolic relationships. It is the ATP stoichiometry that determines the overall thermodynamics of metabolic sequences. By this we mean that the overall reaction mediated by any metabolic pathway is energetically favorable because of its particular ATP stoichiometry. A significant part of the energy released in the highly exergonic reactions of catabolism is captured in ATP synthesis. In turn, energy released upon ATP hydrolysis drives the thermodynamically unfavorable reactions of anabolism. Ultimately, then, the overall thermodynamic efficiency of metabolism is determined through coupling with ATP.

      To illustrate this principle, we must first consider the three types of stoichiometries. The first two are fixed by the laws of chemistry, but the third is unique to living systems and reveals a fundamental difference between the world of chemistry and physics and the world of functional design—that is, the world of living organisms. The fundamental difference is the stoichiometry of ATP coupling.

1. Reaction Stoichiometry

                               C₆H₁₂O₆+6 O₂ ® 6 CO₂+6 H₂O

2. Obligate Coupling Stoichiometry

(a)    C₆H₁₂O₆ + 10 NAD⁺ + 2 [FAD] + 6 H₂O ®
                                                   6 CO₂ + 10 NADH + 10 H⁺ + 2 [FADH₂]

Sequence (a) accounts for the oxidation of glucose via glycolysis and the citric acid cycle. Sequence (b) is the overall equation for electron transport per glucose. The stoichiometry of coupling by the biological e^- carriers, NAD⁺ and FAD, is fixed by the chemistry of electron transfer; each of the coenzymes serves as an e^- pair acceptor. Reduction of each O atom takes an e^- pair. Metabolism must obey these facts of chemistry: biological oxidation of glucose releases 12 e^- pairs, creating an obligate requirement for 12 equivalents of e^- pair acceptors, which transfer the electrons to 12 O atoms.

3. Evolved Coupling Stoichiometries

     C₆H₁₂O₆ + 6 O₂ + 38 ADP + 38 P_i ® 6 CO₂ + 38 ATP + 44 H₂O

The “stoichiometry” of ATP formation, 38 ADP + 38 P_i ® 38 ATP + 38 H₂O, cannot be predicted from any chemical considerations. The value of 38 ATP is an end result of biological adaptation. It is a phenotypic character of organisms—that is, a trait acquired through interaction of heredity and environment over the course of evolution. Like any evolved phenotypic character, this ATP stoichiometry is the result of compromise. As we shall see, more than 38 ATP equivalents are available from the free energy of glucose oxidation, and certainly the biological system could also yield less. Nevertheless, the final trait is one particularly suited to the fitness of the organism.

The Significance of 38 ATP/Glucose in Cellular Respiration

The standard free energy of ATP hydrolysis, -30.5 kJ/mol, is a large negative number. Under physiological conditions of prevailing cellular ATP, ADP, and P_i concentrations, the actual free energy of ATP hydrolysis is probably closer to -50 kJ/mol. The important point is that a change in the ATP “stoichiometry” dramatically affects the K_eq in a coupled reaction. The K_eq of interest here can be calculated from the standard free energy change for glucose oxidation, -2870 kJ/mol. Under cellular conditions where the concentrations of glucose, O₂, and CO₂ may be taken as 10 mM, 0.13 atm, and 0.05 atm, respectively,³ the free energy change, DG°'). The K_eq for the coupling of glucose oxidation is

K_eq=[CO₂]⁶[ATP]ⁿ/[glucose][ADP]ⁿ[P_i]ⁿ[O₂]⁶

The superscript n for ATP, ADP, and P_i denotes that the stoichiometry for ATP is not fixed and could vary. If n = 38 and each ATP “costs” 50 kJ/mol, then the formation of 38 ATP coupled to glucose oxidation will have an overall K_eq of 10¹⁷⁰, an extremely large number! Almost 58 ATP (58 x 50 = 2900 kJ/mol) could be formed from the oxidation of a mole of glucose, if the coupled reactions had an overall SDG of about 0 (K_eq = 1). At the physiological value of n = 38, the astronomically large K_eq for cellular respiration means that under such conditions the combustion of glucose is highly favorable from a thermodynamic point of view. That is, the reaction is emphatically spontaneous and will go essentially to completion. In the other case, where n = 58 and K_eq = 1, the reaction will come to equilibrium before much glucose has been oxidized. Clearly, this would not be advantageous to an organism using glucose for energy, because at equilibrium, no more energy could be obtained even though some glucose was still available for metabolism. The limitation in not being able to utilize all the glucose offsets any advantage resulting from an apparently greater yield of ATP per mole of glucose (58 versus 38).

      The number 38 is not magical. Recall that in eukaryotes, the net yield of ATP per glucose is 30-32, not 38 (Table 21.4). Also, the value of 38 was established a long time ago in evolution, when the prevailing atmospheric conditions and the competitive situation were undoubtedly very different from those today. The significance of this number is that it provides a high yield of ATP for every glucose, yet the yield is still low enough that glucose is metabolized essentially to completion.

Because ATP is coupled with virtually every metabolic process, we can define the coupling coefficient for a process as the moles of ATP produced or consumed per mole of substrate converted (or product formed). Cellular respiration of glucose thus has a coupling coefficient ranging from 30 to 38, depending on cell type. A reaction such as pyruvate kinase has a coupling coefficient of 1 in the physiological direction, whereas the phosphofructokinase and hexokinase reactions, each of which consumes 1 ATP, have coupling coefficients of -1. These coefficients allow us to put a metabolic price on transactions in metabolism. A glucose, then, is maximally “worth” 38 ATP, and it “costs” 1 ATP to make glucose-6-phosphate from glucose via the hexokinase reaction. These points justify the oft-repeated statement that “ATP is the energy currency of the cell.”
      The metabolic unit of exchange is the ATP equivalent, defined as the conversion of ATP to ADP (or ADP to ATP). In some metabolic reactions, such as the activation of fatty acids, ATP is converted to AMP and PP_i. Because two phosphorylation events are required to re-form ATP from AMP, the ATP equivalent for such reactions is -2.

Because of metabolic interrelationships, it is possible to express all metabolic conversions in terms of ATP equivalents and to assign values, or “prices,” to metabolic intermediates. Such expressions are particularly useful for describing the values of common coupling agents such as NADH, NADPH, and FADH₂ in metabolic transactions. The value of NADH is 3 ATP (2.5 in mitochondria)—the number of ATPs formed as a consequence of NADH oxidation in the process of oxidative phosphorylation. In like fashion, FADH₂ oxidation yields 2 ATP (1.5 in mitochondria), so FADH₂ has an ATP value of 2. The metabolic value of NADPH is less obvious. NADPH is not oxidized via electron transport to generate ATP. Also, NAD and NADP do not serve the same purposes in metabolism, despite their great chemical similarity. For the most part, only NADPH couples catabolism to anabolism by carrying the reducing power released in certain catabolic sequences to reductive biosynthetic processes.
      The metabolic value of NADPH is greater than that of NADH. Even though the standard reduction potentials for the NAD⁺/NADH and NADP⁺/NADPH couples are the same, -320 mV (Table 21.1), in the cell, [NAD⁺]>[NADH] and [NADP⁺], [NADPH] (see Chapter 23). Because of these inequalities, the cellular reduction potentials of these two pyridine nucleotides are not equivalent, and under prevailing cellular conditions, the NADP⁺/NADPH couple is a much better electron donating system than the NAD⁺/NADH couple. Because each NADH is worth 2.5-3 ATP, each NADPH can be assigned a value of 3.5-4 ATP.

The fundamental biological purpose of ATP as an energy coupling agent is to drive thermodynamically unfavorable reactions. (As a corollary, metabolic sequences composed of thermodynamically favorable reactions are exploited to drive the phosphorylation of ADP to make ATP.) Nature has devised enzymatic mechanisms that couple unfavorable reactions with ATP hydrolysis. In effect, the energy release accompanying ATP hydrolysis is transmitted to the unfavorable reaction so that the overall free energy change for the coupled process is negative, i.e., favorable. The involvement of ATP serves to alter the free energy change for a reaction; or, to put it another way, the role of ATP is to change the equilibrium ratio of [reactants] to [products] for a reaction.
      Another way of viewing these relationships is to note that, at equilibrium, the concentrations of ADP and P_i will be vastly greater than that of ATP because DG°' for ATP hydrolysis is a large negative number.⁴ However, the cell where this reaction is a equilibrium is a dead cell. The living cell metabolized food molecules to generate ATP. These catabolic ractions proceed with a very large overall decrease in free energy. Kinetic controls over the rates of the catabolic pathways are designed to ensure that the [ATP]/([ADP][P_i]) ratio is maintained very high. The cell, by employing kinetic controls over the rates of metabolic pathways, maintains a very high [ATP]/([ADP][P_i]) ratio so that ATP hydrolysis can serve as the driving force for virtually all biochemical events.

Substrate Cycles Revisited

If the ATP coupling coefficient for a metabolic sequence in one direction differs from the ATP coupling coefficient for the same sequence in the opposite direction, then it is conceivable that the two reactions could constitute a substrate cycle (Chapter 23), where ATP would be hydrolyzed with no net conversion of substrate to product in either direction.
      Reconsider, for example, the ATP-dependent formation of fructose-1,6-bisphosphate by phosphofructokinase (PFK) in glycolysis versus the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose bisphosphatase (FBPase) during gluconeogenesis:             

The ATP coupling coefficient for the PFK reaction is -1; for FBPase it is 0, because ATP is neither produced nor consumed. Now, in living cells, such substrate cycles are usually prevented by kinetic controls that govern the activity of the enzymes catalyzing the reactions in such putative cycles (Figure 28.2). That is, allosteric effectors reciprocally regulate the two enzymes so that only one is significantly active, and ATP energy is not dissipated fruitlessly (Chapter 23).
      The DG°' for the FBPase reaction is -16.7 kJ/mol, so K_eq is about 850. For the PFK reaction, DG°' is -14.2 kJ/mol, so K_{eq = 310. Thus, either reaction is favorable; the one that occurs is determined by the metabolic needs of the cell for either glycolysis or gluconeogenesis.}
      A very important feature of all pairs of oppositely directed metabolic sequences is illustrated by such substrate cycles. The ATP coefficients for the opposing metabolic sequences always differ, and this difference allows both sequences to be thermo-dynamically favorable at all times. The choice as to which sequence operates is decided by metabolic needs as signaled by the changing concentrations of allosteric effectors.

Figure 28.2 · A substrate cycle, in which the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate by phosphofructokinase (PFK) and fructose bisphosphatase (FBPase) is accompanied by a net hydrolysis of ATP. Such substrate cycles, or futile cycles, as they are also called, are generally prevented by reciprocal regulatory controls: AMP and fructose-2,6-bisphosphate stimulate PFK and inhibit FBPase, whereas citrate inhibits PFK and stimulates FBPase.
        In some circumstances, substrate cycles may provide an advantage by generating heat through ATP hydrolysis. For example, a bumblebee must maintain a temperature of 30°C in its thorax in order to fly. If the ambient temperature is only 10°C, simultaneous high activity of both PFK and FBPase releases heat by ATP hydrolysis, permitting flight. In bumblebees, FBPase is not inhibited by AMP—an adaptation that favors heat generation by this substrate cycle.

28.3 · Unidirectionality

Although opposing metabolic sequences may share steps in common (glyco­ly-sis and gluconeogenesis provide a good example), in a functional sense, metabolic pathways can be treated as unidirectional. That is, they are either catabolic or anabolic, not both, and they proceed in only one direction, fulfilling only one physiological purpose—synthesis or degradation. The rates of these opposing metabolic sequences are controlled by allosteric regulators that modulate the activity of regulatory enzymes at key positions in each pathway.
      For this to be possible, both members of any pair of opposing metabolic pathways, such as fatty acid oxidation and fatty acid biosynthesis, must be thermo-dynamically favorable at essentially the same time under the same conditions. Remember that regulation can be imposed only on reactions displaced far from equilibrium. Given an adequate ATP coupling coefficient, essentially any metabolic sequence can be thermodynamically favorable. Here, then, is perhaps the most fundamental, yet most overlooked, role of ATP in metabolism: The ATP coupling coefficient for any metabolic sequence evolved so that the overall equilibrium for the conversion is highly favorable. This role of ATP can be termed its stoichiometric role. By provision of an appropriate number of ATP equivalents in a metabolic pathway, a very large K_eqfor the overall pathway can be established. That is, in thermodynamic terms, the pathway is emphatically unidirectional (the pathway in the reverse direction is highly unfavorable). Thus, metabolic sequences running in opposite directions, such as fatty acid oxidation and fatty acid synthesis (Figure 28.3), have different ATP coupling coefficients, and so both are thermodynamically favorable and each is undirectional.

Figure 28.3 · ATP coupling coefficients for fatty acid oxidation and fatty acid synthesis. Fatty acid oxidation: Each NADH is worth 3 ATP, and each FADH₂ is worth 2, so the ATP coupling coefficient for palmitoyl-CoA oxidation is +35. Fatty acid synthesis: Each NADPH is worth 4 ATP, so the ATP coupling coefficient for palmitoyl-CoA synthesis is -63. The difference in coupling coefficients for these two sequences is 28 ATP equivalents, which guarantees that each sequence is thermodynamically favorable and that the net conversion between palmitoyl-CoA and acetyl-CoA is not determined by equilibrium considerations, but instead by metabolic need.

ellular Respiration Versus CO₂ Fixation—A Vivid Illustration of the Role of ATP Stoichiometry

The most vivid illustration of the stoichiometric role of ATP is provided by the most important pair of oppositely directed metabolic pathways in the biosphere, namely, photosynthetic carbon dioxide fixation and cellular respiration. As pointed out earlier, the ATP coupling coefficient for cellular respiration is 38, and an ATP coupling coefficient of 58 for this metabolic sequence would yield a K_eqclose to 1 (Figure 28.4). Note that the conversion of glucose to CO₂ becomes thermodynamically unfavorable if the ATP coupling coefficient is greater than 58, which of necessity means that the conversion of CO₂ to glucose becomes favorable. The biological fixation of 6 CO₂ to form 1 glucose requires 12 NADPH and 18 ATP, for an ATP coupling coefficient of -66. Given this coupling coefficient, the carbon dioxide fixation pathway has an overall Keq of 10⁷⁶ (Figure 28.4).⁶ Therefore, both cellular respiration and CO₂ fixation have very large overall equilibrium constants. Thus, in thermodynamic terms, both are emphatically favorable because of their respective ATP coupling coefficients. For the same reason, both pathways are unidirectional.

Figure 28.4 · ATP coupling coefficients between the aerobic respiration of glucose to carbon dioxide and the photosynthetic fixation of CO₂ to glucose.

ATP Has Two Metabolic Roles

The role of ATP in metabolism is twofold:

1. It serves in a stoichiometric role to establish large equilibrium constants for metabolic conversions and to give metabolic sequences a unidirectional character. This is the role referred to when we call ATP the energy currency of the cell.

2. ATP also serves as an important allosteric effector in the kinetic regulation of metabolism. Its concentration (relative to those of ADP and AMP) is an index of the energy status of the cell and determines the rates of regulatory enzymes situated at key points in metabolism, such as PFK in glycolysis and FBPase in gluconeogenesis.

Energy transduction and energy storage in the adenylate system—ATP, ADP, and AMP—lie at the very heart of metabolism. The amount of ATP used per minute by a cell is roughly equivalent to the steady-state amount of ATP it contains. Thus, the metabolic lifetime of an ATP molecule is brief. ATP, ADP, and AMP are all important effectors in exerting kinetic control on regulatory enzymes situated at key points in metabolism, so uncontrolled changes in their concentrations could have drastic consequences. The regulation of metabolism by adenylates in turn requires close control of the relative concentrations of ATP, ADP, and AMP. Some ATP-consuming reactions produce ADP; PFK and hexokinase are examples. Others lead to the formation of AMP, as in fatty acid activation by acetyl-CoA synthetases:

      Fatty acid + ATP + coenzyme A ® AMP + PP_i + fatty acyl-CoA

Adenylate kinase (Chapter 19), by catalyzing the reversible phosphorylation of AMP by ATP, provides a direct connection among all three members of the adenylate pool:

The free energy of hydrolysis of a phosphoanhydride bond is essentially the same in ADP and ATP (Chapter 3), and the standard free energy change for this reaction is close to zero (K_eq = 2.27). Because 2 ADP can lead to 1 ATP by this reaction, an ADP is worth  ATP equivalent.

Energy Charge

The role of the adenylate system is to provide phosphoryl groups at high group-transfer potential in order to drive thermodynamically unfavorable reactions. The capacity of the adenylate system to fulfill this role depends on how fully charged it is with phosphoric anhydrides. Energy charge is an index of this capacity:

The denominator represents the total adenylate pool ([ATP] + [ADP] + [AMP]); the numerator is the number of phosphoric anhydride bonds in the pool, two for each ATP and one for each ADP. The factor  normalizes the equation so that energy charge, or E.C., has the range 0 to 1.0. If all the adenylate is in the form of ATP, E.C. = 1.0, and the potential for phosphoryl transfer is maximal. At the other extreme, if AMP is the only adenylate form present, E.C. = 0. It is reasonable to assume that the adenylate kinase reaction is never far from equilibrium in the cell. Then the relative amounts of the three adenine nucleotides are fixed by the energy charge. Figure 28.5 shows the relative changes in the concentrations of the adenylates as energy charge varies from 0 to 1.0.

Figure 28.5 · Relative concentrations of AMP, ADP, and ATP as a function of energy charge. (This graph was constructed assuming that the adenylate kinase reaction is at equilibrium and that DG°' for the reaction is -473 J/mol; K_eq=1.2.)

The Response of Enzymes to Energy Charge

Figure 28.6 · Responses of regulatory enzymes to variation in energy charge. Enzymes in catabolic pathways have as their ultimate metabolic purpose the regeneration of ATP from ADP. Such enzymes show an R pattern of response to energy charge. Enzymes in biosynthetic pathways utilize ATP to drive anabolic reactions; these enzymes follow the U curve in response to energy charge.

Regulatory enzymes typically respond in reciprocal fashion to adenine nucleotides. For example, PFK is stimulated by AMP and inhibited by ATP. If the activities of various regulatory enzymes are examined in vitro as a function of energy charge, an interesting relationship appears. Regulatory enzymes in energy-producing catabolic pathways show greater activity at low energy charge, but the activity falls off abruptly as E.C. approaches 1.0. In contrast, regulatory enzymes of anabolic sequences are not very active at low energy charge, but their activities increase exponentially as E.C. nears 1.0 (Figure 28.6). These contrasting responses are termed R, for ATP-regenerating, and U, for ATP-utilizing. Regulatory enzymes such as PFK and pyrvuate kinase in glycolysis follow the R response curve as E.C. is varied. Note that PFK itself is an ATP-utilizing enzyme, using ATP to phosphorylate fructose-6-phosphate to yield fructose-1,6-bisphosphate. Nevertheless, because PFK acts physiologically as the valve controlling the flux of carbohydrate down the catabolic pathways of cellular respiration that lead to ATP regeneration, it responds as an “R” enzyme to energy charge. Regulatory enzymes in anabolic pathways, such as acetyl-CoA carboxylase, which initiates fatty acid biosynthesis, respond as “U” enzymes.
      The overall purposes of the R and U pathways are diametrically opposite in terms of ATP involvement. Note in Figure 28.6 that the R and U curves intersect at a rather high E.C. value. As E.C. increases past this point, R activities decline precipitously and U activities rise. That is, when E.C. is very high, biosynthesis is accelerated while catabolism diminishes. The consequence of these effects is that ATP is used up faster than it is regenerated, and so E.C. begins to fall. As E.C. drops below the point of intersection, R processes are favored over U. Then, ATP is generated faster than it is consumed, and E.C. rises again. The net result is that the value of energy charge oscillates about a point of steady state (Figure 28.7). The experimental results obtained from careful measurement of the relative amounts of AMP, ADP, and ATP in living cells reveals that normal cells have an energy charge in the neighborhood of 0.85 to 0.88. Maintenance of this steady-state value is one criterion of cell health and normalcy.

Figure 28.7 · The oscillation of energy charge (E.C.) about a steady-state value as a consequence of the offsetting influences of R and U processes on the production and consumption of ATP. As E.C. increases, the rates of R reactions decline, but U reactions go faster. ATP is consumed, and E.C. drops. Below the point of intersection, R processes are more active and U processes are slower, so E.C. recovers. Energy charge oscillates about a steady-state value determined by the intersection point of the R and U curves.

Because energy charge is maintained at a relatively constant value in normal cells, it is not an informative index of cellular capacity to carry out phosphorylation reactions. The relative concentrations of ATP, ADP, and P_i do provide such information, and a function called phosphorylation potential has been defined in terms of these concentrations:

Phosphorylation potential, G, is equal to [ATP]/([ADP][P_i]).

     Note that this expression includes a term for the concentration of inorganic phosphate. [P_i] has substantial influence on the thermodynamics of ATP hydrolysis. In contrast with energy charge, phosphorylation potential varies over a significant range as the actual proportions of ATP, ADP, and P_i in cells vary in response to metabolic state. G ranges from 200 to 800 M^-1, higher levels signifying more ATP and correspondingly greater phosphorylation potential.

28.4 · Metabolism in a Multicellular Organism

In complex multicellular organisms, organ systems have arisen to carry out specific physiological functions. Each organ expresses a repertoire of metabolic pathways that is consistent with its physiological purpose. Such specialization depends on coordination of metabolic responsibilities among organs so that the organism as a whole may thrive. Essentially all cells in animals have the set of enzymes common to the central pathways of intermediary metabolism, especially the enzymes involved in the formation of ATP and the synthesis of glycogen and lipid reserves. Nevertheless, organs differ in the metabolic fuels they prefer as substrates for energy production. Important differences also occur in the ways ATP is used to fulfill the organs’ specialized metabolic functions. To illustrate these relationships, we will consider the metabolic interactions among the major organ systems found in humans: brain, skeletal muscle, heart, adipose tissue, and liver. In particular, the focus will be on energy metabolism in these organs (Figure 28.8).

Figure 28.8 · Metabolic relationships among the major human organs: brain, muscle, heart, adipose tissue, and liver.

The major fuel depots in animals are glycogen in liver and muscle, triacylglycerols (fats) stored in adipose tissue, and protein, most of which is in skeletal muscle. In general, the order of preference for the use of these fuels is the order given: glycogen > triacylglycerol > protein. Nevertheless, the tissues of the body work together to maintain caloric homeostasis, defined as a constant availability of fuels in the blood.

Organ Specializations

Table 28.1 summarizes the energy metabolism of the major human organs.

BRAIN.  The brain has two remarkable metabolic features. First, it has a very high respiratory metabolism. In resting adult humans, 20% of the oxygen consumed is used by the brain, even though it constitutes only 2% or so of body mass. Interestingly, this level of oxygen consumption is independent of mental activity, continuing even during sleep. Second, the brain is an organ with no significant fuel reserves—no glycogen, usable protein, or fat (even in “fatheads”!). Normally, the brain uses only glucose as a fuel and is totally dependent on the blood for a continuous incoming supply. Interruption of glucose supply for even brief periods of time (as in a stroke) can lead to irreversible losses in brain function. The brain uses glucose to carry out ATP synthesis via cellular respiration. High rates of ATP production are necessary to power the plasma membrane Na⁺/K⁺-ATPase so that the membrane potential essential for transmission of nerve impulses is maintained.
      During prolonged fasting or starvation, the body’s glycogen reserves are depleted. Under such conditions, the brain adapts to use b-hydroxybutyrate (Figure 28.9) as a source of fuel, converting it to acetyl-CoA for energy production via the citric acid cycle. b-Hydroxybutyrate (Chapter 24) is formed from fatty acids in the liver. Although the brain cannot use free fatty acids or lipids directly from the blood as fuel, the conversion of these substances to b-hydroxybutyrate in the liver allows the brain to use body fat as a source of energy. The brain’s other potential source of fuel during starvation is glucose obtained from gluconeogenesis in the liver (Chapter 23), using the carbon skeletons of amino acids derived from muscle protein breakdown. The adaptation of the brain to use b-hydroxybutyrate from fat spares protein from degradation until lipid reserves are exhausted.

Figure 28.9 · The structure of b-hydroxybutyrate and its conversion to acetyl-CoA for combustion in the citric acid cycle.

MUSCLE.  Skeletal muscle is responsible for about 30% of the O₂ consumed by the human body at rest. During periods of maximal exertion, skeletal muscle can account for over 90% of the total metabolism. Muscle metabolism is primarily dedicated to the production of ATP as the source of energy for contraction and relaxation. Muscle contraction occurs when a motor nerve impulse causes Ca²⁺ release from specialized endomembrane compartments (the transverse tubules and sarcoplasmic reticulum). Ca²⁺ floods the sarcoplasm (the term denoting the cytosolic compartment of muscle cells), where it binds to troponin C, a regulatory protein, initiating a series of events that culminate in the sliding of myosin thick filaments along actin thin filaments. This mechanical movement is driven by energy released upon hydrolysis of ATP (Chapter 17). The net result is that the muscle shortens. Relaxation occurs when the Ca²⁺ ions are pumped back into the sarcoplasmic reticulum by the action of a Ca²⁺-transporting membrane ATPase. Two Ca²⁺ are translocated per ATP hydrolyzed. The amount of ATP used during relaxation is almost as much as that consumed during contraction.
      Because muscle contraction is an intermittent process that occurs upon demand, muscle metabolism is designed for a demand response. Muscle at rest uses free fatty acids, glucose, or ketone bodies as fuel and produces ATP via oxidative phosphorylation. Resting muscle also contains about 2% glycogen by weight and an amount of phosphocreatine (Figure 28.10) capable of providing enough ATP to power about 4 seconds of exertion. During strenuous exertion, such as a 100-meter sprint, once the phosphocreatine is depleted, muscle relies solely on its glycogen reserves, making the ATP for contraction via glycolysis. In contrast with the citric acid cycle and oxidative phosphorylation pathways, glycolysis is capable of explosive bursts of activity, and the flux of glucose-6-phosphate through this pathway can increase 2000-fold almost instantaneously. The triggers for this activation are Ca²⁺ and the “fight or flight” hormone epinephrine (Chapters 23 and 34). Little interorgan cooperation occurs during strenuous (anaerobic) exercise.

Figure 28.10 · Phosphocreatine serves as a reservoir of ATP-synthesizing potential. When ADP accumulates as a consequence of ATP hydrolysis, creatine kinase catalyzes the formation of ATP at the expense of phosphocreatine. During periods of rest, when ATP levels are restored by oxidative phosphorylation, creatine kinase acts in reverse to restore the phosphocreatine supply.

      Muscle fatigue is the inability of a muscle to maintain power output. During maximum exertion, the onset of fatigue takes only 20 seconds or so. Fatigue is not the result of exhaustion of the glycogen reserves, nor is it a consequence of lactate accumulation in the muscle. Instead, it is caused by a decline in intramuscular pH as protons are generated during glycolysis. (The overall conversion of glucose to two lactate in glycolysis is accompanied by the release of two H¹.) The pH may fall as low as 6.4. It is likely that the decline in PFK activity at low pH leads to a lowered flux of hexose through glycolysis and inadequate ATP levels, causing a feeling of fatigue. One benefit of PFK inhibition is that the ATP remaining is not consumed in the PFK reaction, and the cell is spared the more serious consequences of losing all of its ATP.
      During fasting or excessive activity, skeletal muscle protein is degraded to amino acids so that their carbon skeletons can be used as fuel. Many of the skeletons are converted to pyruvate, which can be transaminated back into alanine for export via the circulation (Figure 28.11). Alanine is carried to the liver, which in turn transaminates it back into pyruvate so that it can serve as a substrate for gluconeogenesis. Although muscle protein can be mobilized as an energy source, it is not efficient for an organism to consume its muscle and lower its overall fitness for survival. Muscle protein represents a fuel of last resort.

Figure 28.11 · The transamination of pyruvate to alanine by glutamate:alanine aminotransferase.

“Brown Fat.”  A specialized type of adipose tissue, so-called brown fat, is found in newborns and hibernating animals. The abundance of mitochondria, with their rich complement of cytochromes, is responsible for the brown color of this fat. As usual, these mitochondria are very active in electron transport-driven proton translocation, but these particular mitochondria contain in their inner membranes a protein, thermogenin, also known as uncoupler protein-1 (Chapter 21), that creates a passive proton channel, permitting the H⁺ ions to re-enter the mitochondrial matrix without generating ATP. Instead, the energy of oxidation is dissipated as heat. Indeed, brown fat is specialized to oxidize fatty acids for heat production rather than ATP synthesis.

LIVER.  The liver serves as the major metabolic processing center in vertebrates. Except for dietary triacylglycerols, which are metabolized principally by adipose tissue, most of the incoming nutrients that pass through the intestinal tract are routed via the portal vein to the liver for processing and distribution. Much of the liver’s activity centers around conversions involving glucose-6-phosphate (Figure 28.12). Glucose-6-phosphate can be converted to glycogen, released as blood glucose, used to generate NADPH and pentoses via the pentose phosphate cycle, or catabolized to acetyl-CoA for fatty acid synthesis or for energy production via oxidative phosphorylation. Most of the liver glucose-6-phosphate arises from dietary carbohydrate, from degradation of glycogen reserves, or from muscle lactate that enters the gluconeogenic pathway.

Figure 28.12 · Metabolic conversions of glucose-6-phosphate in the liver.

      The liver plays an important regulatory role in metabolism by buffering the level of blood glucose. Liver has two enzymes for glucose phosphorylation, hexokinase and glucokinase. Unlike hexokinase, glucokinase has a low affinity for glucose. Its K_m for glucose is high, on the order of 10 mM. When blood glucose levels are high, glucokinase activity augments hexokinase in phos-phorylating glucose as an initial step leading to its storage in glycogen. The major metabolic hormones—epinephrine, glucagon, and insulin—all influence glucose metabolism in the liver to keep blood glucose levels relatively constant (Chapter 34).
      The liver is a major center for fatty acid turnover. When the demand for metabolic energy is high, triacylglycerols are broken down and fatty acids are degraded in the liver to acetyl-CoA to form ketone bodies, which are exported to the heart, brain, and other tissues. If energy demands are low, fatty acids are incorporated into triacylglycerols that are carried to adipose tissue for deposition as fat. Cholesterol is also synthesized in the liver from two-carbon units derived from acetyl-CoA.
      In addition to these central functions in carbohydrate and fat-based energy metabolism, the liver serves other purposes. For example, the liver can use amino acids as metabolic fuels. Amino acids are first converted to their corresponding a-keto acids by aminotransferases. The amino group is excreted after incorporation into urea in the urea cycle. The carbon skeletons of gluconeogenic amino acids can be used for glucose synthesis, whereas those of ketogenic amino acids appear in ketone bodies (see Figure 26.41). The liver is also the principal detoxification organ in the body. The endoplasmic reticulum of liver cells is rich in enzymes that convert biologically active substances such as hormones, poisons, and drugs into less harmful by-products.
      Liver disease leads to serious metabolic derangements, particularly in amino acid metabolism. In cirrhosis, the liver becomes defective in converting NH₄⁺ to urea for excretion, and blood levels of NH₄⁺ rise. Ammonia is toxic to the central nervous system, and coma ensues.

Ethanol Metabolism Alters the NAD⁺/NADH Ratio

Ethanol is metabolized to acetate in the liver by alcohol dehydrogenase and aldehyde dehydrogenase:
                     CH₃CH₂OH + NAD⁺ ® CH₃CHO + NADH + H⁺

                         CH₃CHO + NAD⁺ ® CH₃COO^- + NADH + H⁺

The excess NADH produced inhibits NAD⁺-requiring reactions, such as gluconeogenesis and fatty acid oxidation. Inhibition of fatty acid oxidation causes elevated triacylglycerol levels in the liver. Over time, these triacylglycerols accumulate as fatty deposits. Inhibition of gluconeogenesis leads to buildup of this pathway’s substrate, lactate. Lactic acid accumulation in the blood causes acidosis. A further consequence is that acetaldehyde can form adducts with protein ¾NH₂ groups, which may impair protein function.