13.5 Regulation of Metabolic Pathways in Chapter 13 Introduction to Metabolism

13.5 Regulation of Metabolic Pathways

Metabolic regulation is one of the most remarkable features of living organisms. Of the thousands of enzyme-catalyzed reactions that can take place in a cell, there is probably not one that escapes some form of regulation. This need to regulate every aspect of cellular metabolism becomes clear as one examines the complexity of metabolic reaction sequences. Although it is convenient for the student of biochemistry to divide metabolic processes into “pathways” that play discrete roles in the cell’s economy, no such separation exists in the living cell. Rather, every pathway we discuss in this book is inextricably intertwined with all the other cellular pathways in a multidimensional network of reactions (Fig. 13-28).

A figure shows a three-dimensional meshwork showing interactions between many metabolic pathways. — FIGURE 13-28 Metabolism as a three-dimensional meshwork. A typical eukaryotic cell has the capacity to make about 30,000 different proteins, which catalyze thousands of different reactions involving many hundreds of metabolites, most shared by more than one “pathway.” In this much-simplified overview of metabolic pathways, each dot represents an intermediate compound and each connecting line represents an enzymatic reaction. For a more realistic and far more complex diagram of metabolism, see the online KEGG PATHWAY database (www.genome.ad.jp/kegg/pathway/map/map01100.html); in this interactive map, you can click on each dot to obtain extensive data about the compound and the enzymes for which it is a substrate.

The figure is labeled metabolic pathways and has many complex interactions between lines with dots along them representing metabolic intermediates. The different types of pathways are color-coded. There are a few light red pathways at the upper right, then a central region of many purple pathways and a few blue pathways. A light blue oval is labeled glycan biosynthesis and metabolism. A purple box in the upper right is labeled biodegradation of xenobiotics. There are orange pathways below, with a pink pathway to the left, then more orange beneath an orange oval labeled nucleotide metabolism. A thicker dark blue vertical line running just to the left of this, just to the left of the center, is labeled carbohydrate metabolism and runs down to a circle near the bottom of the figure. There are some purple pathways to its upper left, then a region of bright green pathways above blue pathways above dark green pathways behind an oval labeled lipid metabolism above a dark purple set of pathways at the lower right behind a dark purple oval labeled energy metabolism. There are some pink pathways beneath the dark blue circle near the bottom center, then more orange pathways to the right and some light red and pink pathways to the far right. A red oval to the lower right of the circle is labeled biosynthesis of secondary metabolites. There are many yellow pathways in the region to the right of the circle and behind it, including a circle to the right that extends down behind a pink oval labeled metabolism of cofactors and vitamins. To the upper right of the blue circle, there is an orangish yellow oval labeled amino acid metabolism with an orange oval labeled metabolism of other amino acids to its upper right. There is a light blue region to the upper right of the oval labeled carbohydrate metabolism.

For example, in Chapter 14 we will discuss four possible fates for glucose 6-phosphate in a hepatocyte: breakdown by glycolysis for the production of ATP, breakdown in the pentose phosphate pathway for the production of NADPH and pentose phosphates, use in the synthesis of complex polysaccharides of the extracellular matrix, or hydrolysis to glucose and phosphate to replenish blood glucose. In fact, glucose 6-phosphate has other possible fates in hepatocytes, too; it may, for example, be used to synthesize other sugars, such as glucosamine, galactose, galactosamine, fucose, and neuraminic acid, for use in protein glycosylation, or it may be partially degraded to provide acetyl-CoA for fatty acid and sterol synthesis. And E. coli can use glucose to produce the carbon skeleton of every one of its several thousand types of molecules. When any cell uses glucose 6-phosphate for one purpose, that “decision” affects all the other pathways for which glucose 6-phosphate is a precursor or intermediate: any change in the allocation of glucose 6-phosphate to one pathway affects, directly or indirectly, the flow of metabolites through all the others.

Cells and Organisms Maintain a Dynamic Steady State

The pathways of glucose metabolism provide, in the catabolic direction, the energy essential to oppose the forces of entropy and, in the anabolic direction, biosynthetic precursors and a storage form of metabolic energy. These reactions are so important to survival that very complex regulatory mechanisms have evolved to ensure that metabolites move through each pathway in the correct direction and at the correct rate to match exactly the cell’s or the organism’s changing circumstances. By a variety of mechanisms operating on different time scales, adjustments are made in the rate of metabolite flow through an entire pathway when external circumstances change.

Circumstances do change, sometimes dramatically. The availability of oxygen may decrease due to hypoxia (diminished delivery of oxygen to tissues) or ischemia (diminished flow of blood to tissues). Wound healing requires huge amounts of energy and biosynthetic precursors. The relative proportions of carbohydrate, fat, and protein in the diet vary from meal to meal, and the supply of fuels obtained in the diet is intermittent, requiring metabolic adjustments between meals and during periods of starvation.

Fuels such as glucose enter a cell, and waste products such as ${CO}_{2}$ $CO Subscript 2$ leave, but the mass and the gross composition of a typical cell, organ, or adult animal do not change appreciably over time; cells and organisms exist in a dynamic steady state. For each metabolic reaction in a pathway, the substrate is provided by the preceding reaction at the same rate at which it is converted to product. Thus, although the rate (v) of metabolite flow, or flux, through this step of the pathway may be high and variable, the concentration of substrate, S, remains constant. So, for the two-step reaction

A \overset{v_{1}}{\to} S \overset{v_{2}}{\to} P

$upper A right-arrow Overscript v 1 Endscripts upper S right-arrow Overscript v 2 Endscripts upper P$

when $v_{1} = v_{2}$ $v 1 equals v 2$ , [S] is constant. For example, changes in $v_{1}$ $v 1$ for the entry of glucose from various sources into the blood are balanced by changes in $v_{2}$ $v 2$ for the uptake of glucose from the blood into various tissues, so the concentration of glucose in the blood ([S]) is held nearly constant at 5 mm. This is homeostasis for blood glucose. The failure of homeostatic mechanisms is often at the root of human disease. In diabetes mellitus, for example, the regulation of blood glucose concentration is defective as a result of the lack of or insensitivity to insulin, with profound medical consequences.

In the course of evolution, organisms have acquired a remarkable collection of regulatory mechanisms for maintaining homeostasis at the molecular, cellular, and organismal levels, as reflected in the proportion of genes that encode regulatory machinery. In humans, about 2,500 genes (~12% of all genes) encode regulatory proteins, including a variety of receptors, regulators of gene expression, and more than 500 different protein kinases! In many cases, the regulatory mechanisms overlap: one enzyme is subject to regulation by several different mechanisms.

Both the Amount and the Catalytic Activity of an Enzyme Can Be Regulated

The flux through an enzyme-catalyzed reaction can be modulated by changes in the number of enzyme molecules or by changes in the catalytic activity of each enzyme molecule already present. Such changes occur on time scales from milliseconds to many hours, in response to signals from within or outside the cell. Very rapid allosteric changes in enzyme activity are generally triggered locally, by changes in the local concentration of a small molecule — a substrate of the pathway in which that reaction is a step (say, glucose for glycolysis), a product of the pathway (ATP from glycolysis), or a key metabolite or cofactor (such as NADH) that indicates the cell’s metabolic state. Second messengers (such as cyclic AMP and ${Ca}^{2 +}$ $Ca Superscript 2 plus$ ) generated intracellularly in response to extracellular signals (hormones, cytokines, and so forth) also mediate allosteric regulation, on a slightly slower time scale set by the rate of the signal-transduction mechanism (see Chapter 12).

Extracellular signals (Fig. 13-29, ) may be hormonal (insulin or epinephrine, for example) or neuronal (acetylcholine), or may be growth factors or cytokines. The number of molecules of a given enzyme in a cell is a function of the relative rates of synthesis and degradation of that enzyme. The rate of synthesis can be adjusted by the activation (in response to some outside signal) of a transcription factor (Fig. 13-29, ; described in more detail in Chapter 28). Transcription factors are nuclear proteins that, when activated, bind specific DNA regions (response elements) near a gene’s promoter (its transcriptional starting point) and activate or repress the transcription of that gene, leading to increased or decreased synthesis of the encoded protein. Activation of a transcription factor is sometimes the result of its binding of a specific ligand and sometimes the result of its phosphorylation or dephosphorylation. Each gene is controlled by one or more response elements that are recognized by specific transcription factors. Genes that have several response elements are therefore controlled by several different transcription factors responding to several different signals. Groups of genes encoding proteins that act together, such as the enzymes of glycolysis, often share common response element sequences, so that a single signal, acting through a particular transcription factor, turns all of these genes on and off together.

A figure shows 10 factors that affect enzyme activity: extracellular signals, transcription of specific gene(s), m R N A degradation, m R N A translation on ribosome, protein degradation, sequestration of an enzyme in an organelle, binding of an enzyme to a substrate, binding of an enzyme to a ligand, phosphorylation / dephosphorylation of an enzyme, and interaction of an enzyme with a regulatory protein. — FIGURE 13-29 Factors affecting the activity of enzymes. The total activity of an enzyme can be changed by altering the *number* of its molecules in the cell, or its *effective* activity in a subcellular compartment ( through ), or by modulating the *activity* of existing molecules ( through ), as detailed in the text. An enzyme may be influenced by a combination of such factors.

FIGURE 13-29 Factors affecting the activity of enzymes. The total activity of an enzyme can be changed by altering the *number* of its molecules in the cell, or its *effective* activity in a subcellular compartment ( through ), or by modulating the *activity* of existing molecules ( through ), as detailed in the text. An enzyme may be influenced by a combination of such factors.

A horizontal cell membrane extends from left to right near the top of the figure. A light red rectangular receptor extends across the membrane at the upper left and has a small almost rectangular cutout in its upper right side. There are three pink, almost rectangular structures and three orange spheres above the membrane. An arrow points down from one of the pink structures into the cutout in the receptor, where a similar structure is already present. An arrow from an orange sphere shows that it moves across the membrane. Adjacent text reads, 1 Extracellular signals. An arrow points down from the receptor to a dashed light blue light surrounding a light blue nucleus below. A purple rectangle labeled transcription factor and an orange sphere both join with this arrow. A blue D N A double helix is shown in the nucleus with a purple rectangle attached to its upper left side and an arrow pointing up and to the right to the right of the purple rectangle to a wavy green arrow of m R N A. Adjacent text reads, 2 Transcription of specific gene(s). An arrow points out of the nucleus to a wavy green strand of m R N A below. An arrow points right to many small green pieces beneath text reading, 3 m R N A degradation. An arrow points down from the m R N A to a longer green strand of m R N A with an attached ribosome.The ribosome has a small light blue rectangular piece narrower on the top than on the bottom that overlaps the m R N A with a pink rounded piece below from which a coiled strand is emerging. The left-hand ribosome has the shortest strand and the right-hand ribosome has the longest strand. Text below reads, 4 m R N A translation on ribosome. An arrow points up to a light blue oval labeled enzyme to the upper right. An arrow pointing down to the lower left of the enzyme indicates many small white fragments. Accompanying text reads, 5 protein degradation (ubiquitin; proteasome). An arrow pointing down to the lower right of the enzyme indicates a long purple bar labeled endoplasmic reticulum that extends out of the frame and that contains a gray oval. Text inside the purple bar reads, 6 Enzyme sequestered in subcellular organelles. Paired left- and right-pointing arrows extend from the enzyme to an oval on its right. The right-pointing arrow is joined by a yellow sphere labeled S and the left-pointing arrow shows the loss of a product. The right-hand oval has a yellow sphere labeled S attached to its right side. Text below reads, 7 Enzyme binds substrate yellow sphere labeled S symbol. Paired arrows point from the enzyme to the upper right and back down. The arrow pointing up is joined by a red sphere labeled L and the arrow pointing down shows the loss of a similar red sphere labeled L. To the upper right of the upward arrow, a gray rectangle is shown with a red sphere labeled L attached to its bottom center. Accompanying text reads, 8 Enzyme binds ligand red sphere labeled L symbol (allosteric effector). Similar upward and downward arrows extend up and slightly to the right, where a gray oval is shown with a circle labeled P attached to its left side. The upward arrow is labeled kinase and the downward arrow is labeled phosphatase. Accompanying text reads, 9 Enzyme undergoes phosphorylation / dephosphorylation. Similar upward and downward arrows extend to the upper left of the enzyme, where there is a gray oval with an orange oval bound to its lower side and only partially visible. The upward arrow shows the addition of a similar oval, labeled regulatory protein, and the downward arrow shows the loss of a similar oval. Accompanying text reads, 10 Enzyme undergoes phosphorylation / dephosphorylation.

The stability of messenger RNAs — their resistance to degradation by cellular ribonucleases (Fig. 13-29, ) — varies, and the amount of a given mRNA in the cell is a function of its rates of synthesis and degradation (Chapter 26). The rate at which an mRNA is translated into a protein by ribosomes (Fig. 13-29, ) is also regulated, and depends on several factors described in detail in Chapter 27. Note that an n-fold increase in an mRNA does not always mean an n-fold increase in its protein product.

Once synthesized, protein molecules have a finite lifetime, which may range from minutes to many days (Table 13-8). The rate of protein degradation (Fig. 13-29, ) differs from one protein to another and depends on the conditions in the cell. Some proteins are tagged by the covalent attachment of ubiquitin for degradation in proteasomes, as discussed in Chapter 27 (see, for example, the case of cyclin, in Fig. 12-38). Rapid turnover (synthesis followed by degradation) is energetically expensive, but proteins with a short half-life can reach new steady-state levels much faster than those with a long half-life, and the benefit of this quick responsiveness must balance or outweigh the cost to the cell.

TABLE 13-8 Average Half-Life of Proteins in Mammalian Tissues
Tissue	Average half-life (days)
Liver	0.9
Kidney	1.7
Heart	4.1
Brain	4.6
Muscle	10.7

Yet another way to alter the effective activity of an enzyme is to sequester the enzyme and its substrate in different compartments (Fig. 13-29, ). In muscle, for example, hexokinase cannot act on glucose until the sugar enters the myocyte from the blood, and the rate at which it enters depends on the activity of glucose transporters (see Table 11-1) in the plasma membrane. Within cells, membrane-bounded compartments segregate certain enzymes and enzyme systems, and the transport of substrate across these intracellular membranes may be the limiting factor in enzyme action.

By these several mechanisms for regulating enzyme level, cells can dramatically change their complement of enzymes in response to changes in metabolic circumstances. In vertebrates, liver is the most adaptable tissue; a change from a high-carbohydrate diet to a high-lipid diet, for example, affects the transcription of hundreds of genes and thus the levels of hundreds of proteins. These global changes in gene expression can be quantified in the entire complement of mRNAs (the transcriptome) or protein (proteome) of a cell type or organ, offering great insights into metabolic regulation. The effect of changes in the proteome is often a change in the total ensemble of low molecular weight metabolites, the metabolome (Fig. 13-30). The metabolome of E. coli growing on glucose is dominated by a few classes of metabolites: glutamate (49%); nucleotides (mainly ribonucleoside triphosphates) (15%); intermediates of glycolysis, the citric acid cycle, and the pentose phosphate pathway (central pathways of carbon metabolism) (15%); and redox cofactors and glutathione (9%).

A pie graph shows the relative molar abundance of 103 metabolites produced by italicized E. coli end italics growing on glucose, representing a metabolome. — FIGURE 13-30 The metabolome of *E. coli* growing on glucose. Summary of the relative molar abundance of 103 metabolites as measured by a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). For reference, the absolute concentration of glutamate in living cells is 9.6 mm. [Data from B. D. Bennett et al., *Nature Chem. Biol.* 5:593, 2009, Fig. 1.]

The key shows that blue represents amino acids, green represents nucleotides, orange represents N A D (P) (H), light red represents glutathione, dark gray represents central pathways of carbon metabolism, and light gray represents other intermediates. A blue wedge extending from the 12 o’clock position to the 5 o’clock position is labeled glutamate. The region between the 5 o’clock position and just before the six o’clock position contains five blue wedges that are similar in size, with the first three larger than the last two. In a clockwise direction, these blue wedges are aspartate, valine, glutamine, alanine, and other amino acids. There are six green wedges that extend from just before the six o’clock position to just before the eight o’clock position. The first two are slightly larger than the others and are A T P then U T P. Next is G T P, then d T T P, then a slightly smaller wedge for C T P, then other nucleotides. Next is a thin orange wedge labeled N A D followed by a very thin wedge labeled other redox. A light red region from the eight o’clock position to just before the nine o’clock position consists mostly of a large wedge labeled glutathione followed by a thin wedge labeled glutathione disulfide. A relatively large light gray wedge labeled fructose 1,6-bisphosphate extends from just below the nine o’clock position to the ten o’clock position. Next are three smaller light gray wedges that extend from the ten o’clock position to the eleven o’clock position. These are hexose phosphates, then a narrower wedge labeled 6- phosphogluconate, then other central carbon. The region from the eleven o’clock position to the 12 o’clock position contains a wedge for U D P italicized N end italics acetylglucosamine and a thin wedge for U D P glucose that make up about half the space and then a wedge labeled others that makes up the remaining space. All data are approximate.

Once the regulatory mechanisms that involve protein synthesis and degradation have produced a certain number of molecules of each enzyme in a cell, the activity of those enzymes can be further regulated in several other ways: by the concentration of substrate, the presence of allosteric effectors, covalent modifications, or binding of regulatory proteins — all of which can change the activity of an individual enzyme molecule (Fig. 13-29, to ).

All enzymes are sensitive to the concentration of their substrate(s) (Fig. 13-29, ). Recall that in the simplest case (an enzyme that follows Michaelis-Menten kinetics), the initial rate of the reaction is half-maximal when the substrate is present at a concentration equal to $K_{m}$ $upper K Subscript m$ (that is, when the enzyme is half-saturated with substrate). Activity drops off at lower [S], and when [S] $≪ K_{m}$ $much-less-than upper K Subscript m$ , the reaction rate is linearly dependent on [S].

The relationship between [S] and $K_{m}$ $upper K Subscript m$ is important because intracellular concentrations of substrate are often in the same range as, or lower than, $K_{m}$ $upper K Subscript m$ . The activity of hexokinase, for example, changes with [glucose], and intracellular [glucose] varies with the concentration of glucose in the blood. As we will see, the different forms (isozymes) of hexokinase have different $K_{m}$ $upper K Subscript m$ values and are therefore affected differently by changes in intracellular [glucose], in ways that make sense physiologically. For a number of phosphoryl transfers from ATP, and for redox reactions using NADPH or ${NAD}^{+}$ $NAD Superscript plus$ , the metabolite concentration is well above the $K_{m}$ $upper K Subscript m$ (Fig. 13-31); these cofactors are not likely to be limiting in such reactions.

A graph plots K subscript m end subscript and metabolite concentration for six types of metabolic enzymes. — FIGURE 13-31 Comparison of $K_{m}$ $bold-italic upper K Subscript bold m$ and substrate concentration for some metabolic enzymes. Measured metabolite concentrations for *E. coli* growing on glucose are plotted against the known $K_{m}$ $upper K Subscript m$ for enzymes that consume that metabolite. The solid line is the line of unity (where metabolite concentration $= K_{m}$ $equals upper K Subscript m$ ), and the dashed lines each denote a 10-fold deviation from the line of unity. [Data from B. D. Bennett et al., *Nature Chem. Biol.* 5:593, 2009, Fig. 2.]

FIGURE 13-31 Comparison of $K_{m}$ $bold-italic upper K Subscript bold m$ and substrate concentration for some metabolic enzymes. Measured metabolite concentrations for *E. coli* growing on glucose are plotted against the known $K_{m}$ $upper K Subscript m$ for enzymes that consume that metabolite. The solid line is the line of unity (where metabolite concentration $= K_{m}$ $equals upper K Subscript m$ ), and the dashed lines each denote a 10-fold deviation from the line of unity. [Data from B. D. Bennett et al., *Nature Chem. Biol.* 5:593, 2009, Fig. 2.]

The key shows that a red square represents N A D plus, a blue square represents N A D P H, an orange sphere represents degradation reactions, an orange square represents A T P, a green diamond represents other, and a gray sphere represents central pathways of carbon metabolism. The horizontal axis plots K subscript m end subscript (M) ranging from 10 superscript negative 8 to 10 superscript 0, labeled in increments of 10 superscript 2, and the vertical axis plots metabolite concentration (M) ranging from 10 superscript negative 8 to 10 superscript 0, labeled in increments of 10 superscript 2. A diagonal line runs across the graph from the lower left to the upper right. A dashed diagonal line runs parallel to and above the solid diagonal line from (10 superscript negative 8, 10 superscript negative 7) to (10 superscript negative 1, 10 superscript 0). A second dashed diagonal line runs parallel to and below the solid diagonal line from (10 superscript negative 7, 10 superscript negative 8) to (10 superscript 0, 10 superscript negative 1). There is a single red square at (10 superscript negative 6.5, 10 superscript negative 2.5), then two red squares close together next to a horizontal line of red squares with only a small break in the middle that extends across the upper dashed diagonal line and runs from (10 superscript negative 5, 10 superscript negative 2.5) to (10 superscript negative 3.5, 10 superscript negative 2.5). There is a small horizontal line of blue squares, then a longer horizontal line of blue squares that crosses the upper dashed diagonal line and runs to the solid diagonal line, where it ends just before a single blue square to the right of the solid diagonal line. These blue squares range from (10 superscript negative 5.8, 10 superscript negative 4) to (10 superscript negative 4, 10 superscript negative 4). Orange circles are present just above the dashed diagonal line between the red and blue lines of squares with three just above the red line and many scattered to the right and below in the region bounded by (10 superscript negative 5.5, 10 superscript negative 5.5), (10 superscript negative 4.5, 10 superscript negative 5.58), (10 superscript negative 1.9, 10 superscript negative 4.5), (10 superscript negative 3.9, 10 superscript negative 2.1), and 10 superscript negative 5.2, 10 superscript negative 4). There is a single brown square at (10 superscript negative 7.5, 10 superscript negative 2), then a line of brown squares ranging from (10 superscript negative 5.9, 10 superscript negative 2) across the upper dashed diagonal line to (10 superscript negative 3, 10 superscript negative 2). Green diamonds are abundant in the region bounded by (10 superscript negative 6.5, 10 superscript negative 6), (10 superscript negative 5.5, 10 superscript negative 6), (10 superscript negative 2.5, 10 superscript negative 5.5), and (10 superscript negative 2, 10 superscript negative 1). Gray spheres are abundant in a smaller region ranging from (10 superscript negative 5, 10 superscript negative 5) to (10 superscript negative 2, 10 superscript negative 3), and (10 superscript negative 5.5, 10 superscript negative 2). All data are approximate.

Enzyme activity can be either increased or decreased by an allosteric effector (Fig. 13-29, ; see Fig. 6-37). Allosteric effectors typically convert hyperbolic kinetics to sigmoid kinetics, or vice versa (see Fig. 14-24b, for example). In the steepest part of the sigmoid curve, a small change in the concentration of substrate, or of allosteric effector, can have a large impact on reaction rate. Recall from Chapter 5 (p. 157) that the cooperativity of an allosteric protein can be expressed as a Hill coefficient, with higher coefficients meaning greater cooperativity. For an allosteric enzyme with a Hill coefficient of 4, activity increases from $10 % V_{\max}$ $10 percent-sign upper V Subscript max Baseline$ to $90 % V_{\max}$ $90 percent-sign upper V Subscript max Baseline$ with only a 3-fold increase in [S], compared with the 81-fold rise in [S] needed by an enzyme with no cooperative effects (Hill coefficient of 1; Table 13-9).

TABLE 13-9 Relationship between Hill Coefficient and the Effect of Substrate Concentration on Reaction Rate for Allosteric Enzymes
Hill coefficient $(n_{H})$ $bold left-parenthesis bold-italic n Subscript bold upper H Baseline bold right-parenthesis$	Required change in [S] to increase $V_{0}$ $bold-italic upper V Subscript bold 0$ from 10% to 90% $V_{max}$ $bold-italic upper V Subscript bold max$
0.5	$\times 6,600$ $times 6,600$
1.0	$\times 81$ $times 81$
2.0	$\times 9$ $times 9$
3.0	$\times 4.3$ $times 4.3$
4.0	$\times 3$ $times 3$

Covalent modifications of enzymes or other proteins (Fig. 13-29, ) occur within seconds or minutes of a regulatory signal, typically an extracellular signal. By far the most common modifications are phosphorylation and dephosphorylation (Fig. 13-32); up to half the proteins in a eukaryotic cell are phosphorylated under some circumstances. Phosphorylation by a specific protein kinase may alter the electrostatic features of an enzyme’s active site, cause movement of an inhibitory region of the enzyme out of the active site, alter the enzyme’s interaction with other proteins, or force conformational changes that translate into changes in $V_{max}$ $upper V Subscript max$ or $K_{m}$ $upper K Subscript m$ . For covalent modification to be useful in regulation, the cell must be able to restore the altered enzyme to its original activity state. A family of phosphoprotein phosphatases, at least some of which are themselves under regulation, catalyzes the dephosphorylation of proteins.

A figure shows the reactions involved in protein phosphorylation and dephosphorylation. — FIGURE 13-32 Protein phosphorylation and dephosphorylation. Protein kinases transfer a phosphoryl group from ATP to a Ser, Thr, or Tyr residue in an enzyme or other protein substrate. Protein phosphatases remove the phosphoryl group as $P_{i}$ $upper P Subscript i$ .

FIGURE 13-32 Protein phosphorylation and dephosphorylation. Protein kinases transfer a phosphoryl group from ATP to a Ser, Thr, or Tyr residue in an enzyme or other protein substrate. Protein phosphatases remove the phosphoryl group as $P_{i}$ $upper P Subscript i$ .

Finally, many enzymes are regulated by association with and dissociation from another, regulatory protein (Fig. 13-29, ). For example, the cyclic AMP–dependent protein kinase (PKA; see Fig. 12-6) is inactive until cAMP binding separates catalytic from regulatory (inhibitory) subunits of the enzyme.

These several mechanisms for altering the flux through a step in a metabolic pathway are not mutually exclusive. It is very common for a single enzyme to be regulated at the level of transcription and by both allosteric and covalent mechanisms. The combination provides fast, smooth, effective regulation in response to a very wide array of perturbations and signals.

In the discussions that follow, it is useful to think of changes in enzymatic activity as serving two distinct though complementary roles. We use the term metabolic regulation to refer to processes that serve to maintain homeostasis at the molecular level — to hold some cellular parameter (concentration of a metabolite, for example) at a steady level over time, even as the flow of metabolites through the pathway changes. The term metabolic control refers to a process that leads to a change in the output of a metabolic pathway over time, in response to some outside signal or change in circumstances. The distinction, although useful, is not always easy to make.

Reactions Far from Equilibrium in Cells Are Common Points of Regulation

For some steps in a metabolic pathway the reaction is close to equilibrium, with the cell in its dynamic steady state (Fig. 13-33). The net flow of metabolites through these steps is the small difference between the rates of the forward and reverse reactions, rates that are very similar when a reaction is near equilibrium. Small changes in substrate or product concentration can produce large changes in the net rate, and they can even change the direction of the net flow. We can identify these near-equilibrium reactions in a cell by comparing the mass-action ratio, Q, with the equilibrium constant for the reaction, $K_{eq}^{'}$ $upper K prime Subscript eq$ . Recall that for the reaction $A + B \to C + D$ $upper A plus upper B right-arrow upper C plus upper D$ , $Q = [C] [D] / [A] [B]$ $upper Q equals left-bracket upper C right-bracket left-bracket upper D right-bracket slash left-bracket upper A right-bracket left-bracket upper B right-bracket$ . In practice, when Q and $K_{eq}^{'}$ $upper K prime Subscript eq$ are within 1 to 2 orders of magnitude of each other, the reaction is near equilibrium. This is the case for more than half of the enzymes in the glycolytic pathway, for example (Table 13-10).

A figure shows the rate of 3 steps in a metabolic pathway that converts molecule A to molecule D. Steps 2 and 3 are near equilibrium and step 1 is far from equilibrium. — FIGURE 13-33 Near-equilibrium and nonequilibrium steps in a metabolic pathway. Steps and of this pathway are near equilibrium in the cell; for each step, the rate (V) of the forward reaction is only slightly greater than the reverse rate, so the net forward rate (10) is relatively low and the free-energy change, ∆G, is close to zero. An increase in [C] or [D] can reverse the direction of these steps. Step is maintained in the cell far from equilibrium; its forward rate greatly exceeds its reverse rate. The net rate of step (10) is much larger than the reverse rate (0.01) and is identical to the net rates of steps and when the pathway is operating in the steady state. Step has a large, negative ∆G.

FIGURE 13-33 Near-equilibrium and nonequilibrium steps in a metabolic pathway. Steps and of this pathway are near equilibrium in the cell; for each step, the rate (V) of the forward reaction is only slightly greater than the reverse rate, so the net forward rate (10) is relatively low and the free-energy change, ∆G, is close to zero. An increase in [C] or [D] can reverse the direction of these steps. Step is maintained in the cell far from equilibrium; its forward rate greatly exceeds its reverse rate. The net rate of step (10) is much larger than the reverse rate (0.01) and is identical to the net rates of steps and when the pathway is operating in the steady state. Step has a large, negative ∆G.

TABLE 13-10 Equilibrium Constants, Mass-Action Ratios, and Free-Energy Changes for Enzymes of Carbohydrate Metabolism
Enzyme	$K_{eq}^{'}$ $bold-italic upper K prime Subscript eq$	Mass-action ratio, Q		Reaction near equilibrium in vivo?^a	$Δ G^{'} °$ $upper Delta upper G prime degree$ (kJ/mol)	ΔG (kJ/mol) in heart
Enzyme	$K_{eq}^{'}$ $bold-italic upper K prime Subscript eq$	Liver	Heart	Reaction near equilibrium in vivo?^a	$Δ G^{'} °$ $upper Delta upper G prime degree$ (kJ/mol)	ΔG (kJ/mol) in heart
Hexokinase	$1 \times 10^{3}$ $1 times 10 cubed$	$2 \times 10^{- 2}$ $2 times 10 Superscript negative 2$	$8 \times 10^{- 2}$ $8 times 10 Superscript negative 2$	No	$- 17$ $negative 17$	$- 27$ $negative 27$
PFK-1	$1.0 \times 10^{3}$ $1.0 times 10 cubed$	$9 \times 10^{- 2}$ $9 times 10 Superscript negative 2$	$3 \times 10^{- 2}$ $3 times 10 Superscript negative 2$	No	$- 14$ $negative 14$	$- 23$ $negative 23$
Aldolase	$1.0 \times 10^{- 4}$ $1.0 times 10 Superscript negative 4$	$1.2 \times 10^{- 6}$ $1.2 times 10 Superscript negative 6$	$9 \times 10^{- 6}$ $9 times 10 Superscript negative 6$	Yes	$+ 24$ $plus 24$	$- 6.0$ $negative 6.0$
Triose phosphate isomerase	$4 \times 10^{- 2}$ $4 times 10 Superscript negative 2$	— ^b	$2.4 \times 10^{- 1}$ $2.4 times 10 Superscript negative 1$	Yes	$+ 7.5$ $plus 7.5$	$+ 3.8$ $plus 3.8$
Glyceraldehyde 3-phosphate dehydrogenase + phosphoglycerate kinase	$2 \times 10^{3}$ $2 times 10 cubed$	$6 \times 10^{2}$ $6 times 10 squared$	9.0	Yes	$- 13$ $negative 13$	$+ 3.5$ $plus 3.5$
Phosphoglycerate mutase	$1 \times 10^{- 1}$ $1 times 10 Superscript negative 1$	$1 \times 10^{- 1}$ $1 times 10 Superscript negative 1$	$1.2 \times 10^{- 1}$ $1.2 times 10 Superscript negative 1$	Yes	$+ 4.4$ $plus 4.4$	$+ 0.6$ $plus 0.6$
Enolase	3	2.9	1.4	Yes	$- 3.2$ $negative 3.2$	$- 0.5$ $negative 0.5$
Pyruvate kinase	$2 \times 10^{4}$ $2 times 10 Superscript 4$	$7 \times 10^{- 1}$ $7 times 10 Superscript negative 1$	40	No	$- 31$ $negative 31$	$- 17$ $negative 17$
Phosphohexose isomerase	$4 \times 10^{- 1}$ $4 times 10 Superscript negative 1$	$3.1 \times 10^{- 1}$ $3.1 times 10 Superscript negative 1$	$2.4 \times 10^{- 1}$ $2.4 times 10 Superscript negative 1$	Yes	$+ 2.2$ $plus 2.2$	$- 1.4$ $negative 1.4$
Pyruvate carboxylase + PEP carboxykinase	7	$1 \times 10^{- 3}$ $1 times 10 Superscript negative 3$	— ^b	No	$- 5.0$ $negative 5.0$	$- 23$ $negative 23$
Glucose 6-phosphatase	$8.5 \times 10^{2}$ $8.5 times 10 squared$	$1.2 \times 10^{2}$ $1.2 times 10 squared$	— ^b	Yes	$- 17$ $negative 17$	$- 5.0$ $negative 5.0$
Data for $K_{eq}^{'}$ $upper K prime Subscript eq$ and Q from E. A. Newsholme and C. Start, Regulation in Metabolism, pp. 97, 263, Wiley Press, 1973. ∆G and $Δ G^{'} °$ $upper Delta upper G prime degree$ were calculated from these data. ^aFor simplicity, any reaction for which the absolute value of the calculated ∆G is less than 6 is considered near equilibrium. ^bData not available.

Other reactions are far from equilibrium in the cell. For example, $K_{eq}^{'}$ $upper K prime Subscript eq$ for the phosphofructokinase-1 (PFK-1) reaction is about 1,000, but Q ([fructose 1,6-bisphosphate][ADP]/[fructose 6-phosphate][ATP]) in a hepatocyte in the steady state is about 0.1 (Table 13-10). It is because the reaction is so far from equilibrium in the cell that the process is exergonic under cellular conditions and tends to go in the forward direction. The reaction is held far from equilibrium because, under prevailing cellular conditions of substrate, product, and effector concentrations, the rate of conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is limited by the activity of PFK-1. PFK-1 activity itself is limited by the number of PFK-1 molecules present and by the actions of allosteric effectors. Thus, the net forward rate of the enzyme-catalyzed reaction is equal to the net flow of glycolytic intermediates through other steps in the pathway, and the reverse flow through PFK-1 remains near zero.

The cell cannot allow reactions with large equilibrium constants to reach equilibrium. If [fructose 6-phosphate], [ATP], and [ADP] in the cell were held at typical levels (low millimolar concentrations) and the PFK-1 reaction were allowed to reach equilibrium by an increase in [fructose 1,6-bisphosphate], the concentration of fructose 1,6-bisphosphate would rise into the molar range, wreaking osmotic havoc on the cell. Consider another case: if the reaction $ATP \to ADP + P_{i}$ $ATP right-arrow ADP plus upper P Subscript i Baseline$ were allowed to approach equilibrium in the cell, the actual free-energy change (ΔG) for that reaction ( $Δ G_{p}$ $upper Delta upper G Subscript p Baseline$ ; see Worked Example 13-2, p. 480) would approach zero, and ATP would lose the high phosphoryl group transfer potential that makes it valuable to the cell. It is therefore essential that enzymes catalyzing ATP breakdown and other highly exergonic reactions in a cell be sensitive to regulation, so that when metabolic changes are forced by external circumstances, the flow through these enzymes will be adjusted to ensure that [ATP] remains far above its equilibrium level. When such metabolic changes occur, the activities of enzymes in all interconnected pathways adjust to keep these critical steps away from equilibrium. Thus, not surprisingly, many enzymes that catalyze highly exergonic reactions are subject to a variety of subtle regulatory mechanisms. The multiplicity of these adjustments is so great that we cannot predict by examining the properties of any one enzyme in a pathway whether that enzyme has a strong influence on net flow through the entire pathway.

Adenine Nucleotides Play Special Roles in Metabolic Regulation

After the protection of its DNA from damage, perhaps nothing is more important to a cell than maintaining a constant supply and concentration of ATP. Many ATP-using enzymes have $K_{m}$ $upper K Subscript m$ values between 0.1 and 1 mm, and the ATP concentration in a typical cell is about 5 to 10 mm (Fig. 13-31). If [ATP] were to drop significantly, these enzymes would be less than fully saturated by their substrate (ATP), and the rates of hundreds of reactions that involve ATP would decrease (Fig. 13-34); the cell would probably not survive this kinetic effect on so many reactions.

A graph plots initial velocity against A T P concentration. The curve is labeled with one-half V subscript max and ends below a dashed horizontal line labeled V subscript max. — FIGURE 13-34 Effect of ATP concentration on the initial reaction velocity of a typical ATP-dependent enzyme. These experimental data yield a $K_{m}$ $upper K Subscript m$ for ATP of 5 mm. The concentration of ATP in animal tissues is ~5 mm.

FIGURE 13-34 Effect of ATP concentration on the initial reaction velocity of a typical ATP-dependent enzyme. These experimental data yield a $K_{m}$ $upper K Subscript m$ for ATP of 5 mm. The concentration of ATP in animal tissues is ~5 mm.

The horizontal axis is labeled A T P concentration [m M] and ranges from below 5 to above 45, labeled in increments of 5, and the vertical axis is labeled initial velocity, V (arbitrary units). A dashed horizontal line labeled V subscript max extends from just above one-quarter of the height of the vertical axis. The curve begins at the origin and rises relatively quickly at first, then begins to slow at 2.5 on the horizontal axis and almost one-quarter of the vertical axis. IT rises more slowly through a point labeled one-half V subscript max at 5 on the horizontal axis and almost half the height of the vertical axis. A dashed horizontal line extends from this point to the vertical axis and a dashed vertical line extends from this point to the horizontal axis. The curve begins to level off until it is almost horizontal by the time it reaches 40 on the horizontal axis just below the dashed line labeled V subscript max. All data are approximate.

There is also an important thermodynamic effect of lowered [ATP]. Because ATP is converted to ADP or AMP when “spent” to accomplish cellular work, the [ATP]/[ADP] ratio profoundly affects all reactions that employ these cofactors. The same is true for other important cofactors, such as ${NADH/NAD}^{+}$ $NADH slash NAD Superscript plus$ and ${NADPH/NADP}^{+} .$ $NADPH slash NADP Superscript plus Baseline period$ For example, consider the reaction catalyzed by hexokinase:

ATP + glucose \overset{}{\to} ADP + glucose 6 -phosphate

$ATP plus glucose right-arrow Overscript Endscripts ADP plus glucose 6 hyphen phosphate$

K_{eq}^{'} = \frac{{[ADP]}_{eq} {[glucose 6 -phosphate]}_{eq}}{[ATP]_{eq} [glucose]_{eq}} = 2 \times 10^{3}

$upper K prime Subscript eq Baseline equals StartFraction left-bracket ADP right-bracket Subscript eq Baseline left-bracket glucose 6 hyphen phosphate right-bracket Subscript eq Baseline Over left-bracket ATP right-bracket Subscript eq Baseline left-bracket glucose right-bracket Subscript eq Baseline EndFraction equals 2 times 10 cubed$

Note that this expression holds true only when reactants and products are at their equilibrium concentrations, where $Δ G = 0$ $upper Delta upper G equals 0$ . At any other set of concentrations, $Δ G$ $upper Delta upper G$ is not zero. Recall (from Section 13.1) that the ratio of products to substrates (the mass-action ratio, Q) determines the magnitude and sign of $Δ G$ $upper Delta upper G$ and therefore the driving force, $Δ G$ $upper Delta upper G$ , of the reaction:

Δ G = Δ G^{'} ° + R T ln \frac{[ADP] [glucose 6 -phosphate]}{[ATP] [glucose]}

$upper Delta upper G equals upper Delta upper G prime degree plus upper R upper T ln StartFraction left-bracket ADP right-bracket left-bracket glucose 6 hyphen phosphate right-bracket Over left-bracket ATP right-bracket left-bracket glucose right-bracket EndFraction$

Because an alteration of this driving force profoundly influences every reaction that involves ATP, organisms have evolved under strong pressure to develop regulatory mechanisms responsive to the [ATP]/[ADP] ratio.

AMP concentration is an even more sensitive indicator of a cell’s energetic state than is [ATP]. Normally, cells have a far higher concentration of ATP (5 to 10 mm) than of AMP (<0.1 mm). When some process (say, muscle contraction) consumes ATP, AMP is produced in two steps. First, hydrolysis of ATP produces ADP, then the reaction catalyzed by adenylate kinase produces AMP:

2 ADP \overset{}{\to} AMP + ATP

$2 ADP right-arrow Overscript Endscripts AMP plus ATP$

If ATP is consumed such that its concentration drops 10%, the relative increase in [AMP] is much greater than that of [ADP] (Table 13-11). It is not surprising, therefore, that many regulatory processes are keyed to changes in [AMP]. Probably the most important mediator of regulation by AMP is AMP-activated protein kinase (AMPK), which responds to an increase in [AMP] by phosphorylating key proteins and thus regulating their activities. (AMPK is not to be confused with the cyclic AMP–dependent protein kinase PKA; see Section 12.2.) The rise in [AMP] may be caused by a reduced nutrient supply or by increased physical exercise. AMP activates AMPK allosterically, which increases glucose transport and activates glycolysis and fatty acid oxidation, while suppressing energy-requiring processes such as the synthesis of glycogen, fatty acids, cholesterol, and protein. All of the changes effected by AMPK serve to raise [ATP] and lower [AMP]. In Chapter 23, we discuss the role of AMPK in balancing anabolism and catabolism in the whole organism.

TABLE 13-11 Relative Changes in [ATP] and [AMP] When ATP Is Consumed
Adenine nucleotide	Concentration before ATP depletion (mm)	Concentration after ATP depletion (mm)	Relative change
ATP	5.0	4.5	10%
ADP	1.0	1.0	0
AMP	0.1	0.6	600%

SUMMARY 13.5 Regulation of Metabolic Pathways

In a metabolically active cell in a steady state, intermediates are formed and consumed at equal rates. When a transient perturbation alters the rate of formation or consumption of a metabolite, compensating changes in enzyme activities return the system to the steady state.
Cells regulate their metabolism by a variety of mechanisms over a time scale ranging from less than a millisecond to days, either by changing the activity of existing enzyme molecules or by changing the number of molecules of a specific enzyme.
Various signals activate or inactivate transcription factors, which act in the nucleus to regulate gene expression. Changes in the transcriptome lead to changes in the proteome, and ultimately in the metabolome of a cell or tissue.
In multistep processes such as glycolysis, certain reactions are essentially at equilibrium in the steady state; the rates of these reactions rise and fall with substrate concentration. Other reactions are far from equilibrium; these steps are typically the points of regulation of the overall pathway.
Regulatory mechanisms maintain nearly constant levels of key metabolites such as ATP and NADH in cells and glucose in the blood, while matching the use or production of glucose to the organism’s changing needs.
The levels of ATP and AMP are a sensitive reflection of a cell’s energy status, and when the [ATP]/[AMP] ratio decreases, the AMP-activated protein kinase (AMPK) triggers a variety of cellular responses to raise [ATP] and lower [AMP].