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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 16.2The Glycolytic Pathway Is Tightly Controlled

The flux through the glycolytic pathway must be adjusted in response to conditions both inside and outside the cell. The rate of conversion of glucose into pyruvate is regulated to meet two major cellular needs: (1) the production of ATP, generated by the degradation of glucose, and (2) the provision of building blocks for synthetic reactions, such as the formation of fatty acids. In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites of control. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible; hence, these enzymes would be expected to have regulatory as well as catalytic roles. In fact, each of them serves as a control site. Their activities are regulated by the reversible binding of allosteric effectors or by covalent modification. In addition, the amounts of these important enzymes are varied by the regulation of transcription to meet changing metabolic needs. The time required for reversible allosteric control, regulation by phosphorylation, and transcriptional control is typically in milliseconds, seconds, and hours, respectively.

16.2.1. Phosphofructokinase Is the Key Enzyme in the Control of Glycolysis

Phosphofructokinase is the most important control element in the mammalian glycolytic pathway (Figure 16.16). High levels of ATP allosterically inhibit the enzyme in the liver (a 340-kd tetramer), thus lowering its affinity for fructose 6-phosphate. A high concentration of ATP converts the hyperbolic binding curve of fructose 6-phosphate into a sigmoidal one (Figure 16.17). ATP elicits this effect by binding to a specific regulatory site that is distinct from the catalytic site. AMP reverses the inhibitory action of ATP, and so the activity of the enzyme increases when the ATP/AMP ratio is lowered. In other words, glycolysis is stimulated as the energy charge falls. A fall in pH also inhibits phosphofructokinase activity. The inhibition of phosphofructokinase by H+ prevents excessive formation of lactic acid (Section 16.1.9) and a precipitous drop in blood pH (acidosis).

Figure 16.16. Structure of Phosphofructokinase.

Figure 16.16

Structure of Phosphofructokinase. Image mouse.jpg Phosphofructokinase in the liver is a tetramer of four identical subunits. The positions of the catalytic and allosteric sites are indicated.

Figure 16.17. Allosteric Regulation of Phosphofructokinase.

Figure 16.17

Allosteric Regulation of Phosphofructokinase. A high level of ATP inhibits the enzyme by decreasing its affinity for fructose 6-phosphate. AMP diminishes and citrate enhances the inhibitory effect of ATP.

Why is AMP and not ADP the positive regulator of phosphofructokinase? When ATP is being utilized rapidly, the enzyme adenylate kinase (Section 9.4) can form ATP from ADP by the following reaction:

Image ch16e9.jpg

Thus, some ATP is salvaged from ADP, and AMP becomes the signal for the low-energy state. Moreover, the use of AMP as an allosteric regulator provides an especially sensitive control. We can understand why by considering, first, that the total adenylate pool ([ATP], [ADP], [AMP]) in a cell is constant over the short term and, second, that the concentration of ATP is greater than that of ADP and the concentration of ADP is, in turn, greater than that of AMP. Consequently, small-percentage changes in [ATP] result in larger-percentage changes in the concentrations of the other adenylate nucleotides. This magnification of small changes in [ATP] to larger changes in [AMP] leads to tighter control by increasing the range of sensitivity of phosphofructokinase.

Glycolysis also furnishes carbon skeletons for biosyntheses, and so a signal indicating whether building blocks are abundant or scarce should also regulate phosphofructokinase. Indeed, phosphofructokinase is inhibited by citrate, an early intermediate in the citric acid cycle (Section 17.1.3). A high level of citrate means that biosynthetic precursors are abundant and additional glucose should not be degraded for this purpose. Citrate inhibits phosphofructokinase by enhancing the inhibitory effect of ATP.

In 1980, fructose 2,6-bisphosphate (F-2,6-BP) was identified as a potent activator of phosphofructokinase. Fructose 2,6-bisphosphate activates phosphofructokinase by increasing its affinity for fructose 6-phosphate and diminishing the inhibitory effect of ATP (Figure 16.18). In essence, Fructose 2,6-bisphosphate is an allosteric activator that shifts the conformational equilibrium of this tetrameric enzyme from the T state to the R state.

Image ch16fu24.jpg

Figure 16.18. Activation of Phosphofructokinase by Fructose 2,6-Bisphosphate.

Figure 16.18

Activation of Phosphofructokinase by Fructose 2,6-Bisphosphate. (A) The sigmoidal dependence of velocity on substrate concentration becomes hyperbolic in the presence of 1 μM fructose 2,6-bisphosphate. (B) ATP, acting as a substrate, initially (more...)

16.2.2. A Regulated Bifunctional Enzyme Synthesizes and Degrades Fructose 2,6 -bisphosphate

How is the concentration of fructose 2,6-bisphosphate appropriately controlled? Two enzymes regulate the concentration of this important regulator of glycolysis by phosphorylating fructose 6-phosphate and dephosphorylating fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is formed in a reaction catalyzed by phosphofructokinase 2 (PFK2), a different enzyme from phosphofructokinase. Fructose 2,6-bisphosphate is hydrolyzed to fructose 6-phosphate by a specific phosphatase, fructose bisphosphatase 2 (FBPase2). The striking finding is that both PFK2 and FBPase2 are present in a single 55-kd polypeptide chain (Figure 16.19). This bifunctional enzyme contains an N-terminal regulatory domain, followed by a kinase domain and a phosphatase domain. PFK2 resembles adenylate kinase in having a P-loop NTPase domain (Sections 9.4.1 and 9.4.3), whereas FBPase2 resembles phosphoglycerate mutase (Section 16.1.7). Recall that the mutase is essentially a phosphatase. In the bifunctional enzyme, the phosphatase activity evolved to become specific for F-2,6-BP. The bifunctional enzyme itself probably arose by the fusion of genes encoding the kinase and phosphatase domains.

Figure 16.19. Domain Structure of the Bifunctional Enzyme Phosphofructokinase 2.

Figure 16.19

Domain Structure of the Bifunctional Enzyme Phosphofructokinase 2. Image mouse.jpg The kinase domain (purple) is fused to the phosphatase domain (red). The kinase domain is a P-loop NTP hydrolase domain, as indicated by the purple shading (Section 9.4.4). The bar represents (more...)

The bifunctional enzyme exists in five isozymic forms (isoforms) that differ in size and kinetics as well as immunological and regulatory properties. Recall that isoenzymes, or isozymes, have essentially the same architectural plan and catalytic properties but differ in how they are regulated. The L isoform, which predominates in the liver, and the M isoform, found in muscle are generated by alternative splicing (Section 28.3.6) of the transcription product of a single gene. The L isoform helps to maintain blood-glucose homeostasis. In the liver, the concentration of fructose 6-phosphate rises when blood-glucose concentration is high, and the abundance of fructose 6-phosphate accelerates the synthesis of F-2,6-BP. Hence, an abundance of fructose 6-phosphate leads to a higher concentration of F-2,6-BP, which in turn stimulates phosphofructokinase. Such a process is called feedforward stimulation. What controls whether PFK2 or FBPase2 dominates the bifunctional enzyme's activities in the liver? The activities of PFK2 and FBPase2 are reciprocally controlled by phosphorylation of a single serine residue. When glucose is scarce, a rise in the blood level of the hormone glucagon triggers a cyclic AMP cascade, through its 7TM receptor and Gαs (Section 15.1), leading to the phosphorylation of this bifunctional enzyme by protein kinase A (Figure 16.20). This covalent modification activates FBPase2 and inhibits PFK2, lowering the level of F-2,6-BP. Thus, glucose metabolism by the liver is curtailed. Conversely, when glucose is abundant, the enzyme loses its attached phosphate group. This covalent modification activates PFK2 and inhibits FBPase2, raising the level of F-2,6-BP and accelerating glycolysis. This coordinated control is facilitated by the location of the kinase and phosphatase domains on the same polypeptide chain as the regulatory domain. We shall return to this elegant switch when we consider the integration of carbohydrate metabolism (Section 16.4).

Figure 16.20. Control of the Synthesis and Degradation of Fructose 2,6-Bisphosphate.

Figure 16.20

Control of the Synthesis and Degradation of Fructose 2,6-Bisphosphate. A low blood-glucose level as signaled by glucagon leads to the phosphorylation of the bifunctional enzyme and hence to a lower level of fructose 2,6-bisphosphate, slowing glycolysis. (more...)

16.2.3. Hexokinase and Pyruvate kinase Also Set the Pace of Glycolysis

Phosphofructokinase is the most prominent regulatory enzyme in glycolysis, but it is not the only one. Hexokinase, the enzyme catalyzing the first step of glycolysis, is inhibited by its product, glucose 6-phosphate. High concentrations of this molecule signal that the cell no longer requires glucose for energy, for storage in the form of glycogen, or as a source of biosynthetic precursors, and the glucose will be left in the blood. For example, when phosphofructokinase is inactive, the concentration of fructose 6-phosphate rises. In turn, the level of glucose 6-phosphate rises because it is in equilibrium with fructose 6-phosphate. Hence, the inhibition of phosphofructokinase leads to the inhibition of hexokinase. However, the liver, in keeping with its role as monitor of blood-glucose levels, possesses a specialized isozyme of hexokinase called glucokinase that is not inhibited by glucose 6-phosphate. Glucokinase phosphorylates glucose only when it is abundant because it has about a 50-fold affinity for glucose than does hexokinase. The role of glucokinase is to provide glucose 6-phosphate for the synthesis of glycogen, a storage form of glucose (Section 21.4), and for the formation of fatty acids (Section 22.1). The low glucose affinity of glucokinase in the liver gives the brain and muscles first call on glucose when its supply is limited, whereas it ensures that glucose will not be wasted when it is abundant.

Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis? The reason becomes evident on noting that glucose 6-phosphate is not solely a glycolytic intermediate. Glucose 6-phosphate can also be converted into glycogen or it can be oxidized by the pentose phosphate pathway (Section 20.3) to form NADPH. The first irreversible reaction unique to the glycolytic pathway, the committed step, (Section 10.2), is the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Thus, it is highly appropriate for phosphofructokinase to be the primary control site in glycolysis. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most important control element in the pathway.

Pyruvate kinase, the enzyme catalyzing the third irreversible step in glycolysis, controls the outflow from this pathway. This final step yields ATP and pyruvate, a central metabolic intermediate that can be oxidized further or used as a building block. Several isozymic forms of pyruvate kinase (a tetramer of 57-kd subunits) encoded by different genes are present in mammals: the L type predominates in liver, and the M type in muscle and brain. The L and M forms of pyruvate kinase have many properties in common. Both bind phosphoenolpyruvate cooperatively. Fructose 1,6-bisphosphate, the product of the preceding irreversible step in glycolysis, activates both isozymes to enable them to keep pace with the oncoming high flux of intermediates. ATP allosterically inhibits both the L and the M forms of pyruvate kinase to slow glycolysis when the energy charge is high. Finally, alanine (synthesized in one step from pyruvate, Section 24.2.2) also allosterically inhibits the pyruvate kinases—in this case, to signal that building blocks are abundant.

The isozymic forms differ in their susceptibility to covalent modification. The catalytic properties of the L form—but not of the M form—are also controlled by reversible phosphorylation (Figure 16.21). When the blood-glucose level is low, the glucagon-triggered cyclic AMP cascade (Section 15.1.5) leads to the phosphorylation of pyruvate kinase, which diminishes its activity. These hormone-triggered phosphorylations, like that of the bifunctional enzyme controlling the levels of fructose 2,6-bisphosphate, prevent the liver from consuming glucose when it is more urgently needed by brain and muscle (Section 30.3). We see here a clear-cut example of how isoenzymes contribute to the metabolic diversity of different organs. We will return to the control of glycolysis after considering gluconeogenesis.

Figure 16.21. Control of the Catalytic Activity of Pyruvate Kinase.

Figure 16.21

Control of the Catalytic Activity of Pyruvate Kinase. Pyruvate kinase is regulated by allosteric effectors and covalent modification.

16.2.4. A Family of Transporters Enables Glucose to Enter and Leave Animal Cells

Several glucose transporters mediate the thermodynamically downhill movement of glucose across the plasma membranes of animal cells. Each member of this protein family, named GLUT1 to GLUT5, consists of a single polypeptide chain about 500 residues long (Table 16.4). The common structural theme is the presence of 12 transmembrane segments (Figure 16.22).

Table 16.4. Family of glucose transporters.

Table 16.4

Family of glucose transporters.

Figure 16.22. Model of a Mammalian Glucose Transporter.

Figure 16.22

Model of a Mammalian Glucose Transporter. The hydrophobicity profile of the protein indicates 12 transmembrane α helices. [From M. Muekler, C. Caruso, S. A. Baldwin, M. Panico, M. Blench, H. R. Morris, W. J. Allard, G. E. Lienhard, and H. F. Lodish. (more...)

The members of this family have distinctive roles:


GLUT1 and GLUT3, present in nearly all mammalian cells, are responsible for basal glucose uptake. Their KM value for glucose is about 1 mM, significantly less than the normal serum-glucose level, which typically ranges from 4 mM to 8 mM. Hence, GLUT1 and GLUT3 continually transport glucose into cells at an essentially constant rate.


GLUT2, present in liver and pancreatic β cells, is distinctive in having a very high KM value for glucose (15–20 mM). Hence, glucose enters these tissues at a biologically significant rate only when there is much glucose in the blood. The pancreas can thereby sense the glucose level and accordingly adjust the rate of insulin secretion. Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat (Section 30.3). The high KM value of GLUT2 also ensures that glucose rapidly enters liver cells only in times of plenty.


GLUT4, which has a KM value of 5 mM, transports glucose into muscle and fat cells. The presence of insulin, which signals the fed state, leads to a rapid increase in the number of GLUT4 transporters in the plasma membrane. Hence, insulin promotes the uptake of glucose by muscle and fat. The amount of this transporter present in muscle membranes increases in response to endurance exercise training.


GLUT5, present in the small intestine, functions primarily as a fructose transporter.

Image tree.jpg This family of transporters vividly illustrates how isoforms of a single protein can significantly shape the metabolic character of cells and contribute to their diversity and functional specialization. The transporters are members of a superfamily of transporters called the major facilitator (MF) superfamily. Members of this family transport sugars in organisms as diverse as E. coli, Trypansoma brucei (a parasitic protozoan that causes sleeping sickness), and human beings. An elegant solution to the problem of fuel transport evolved early and has been tailored to meet the needs of different organisms and even different tissues.

16.2.5. Cancer and Glycolysis

Image caduceus.jpg It has been known for decades that tumors display enhanced rates of glucose uptake and glycolysis. We now know that these enhanced rates of glucose processing are not fundamental to the development of cancer, but we can ask what selective advantage they might confer on cancer cells.

Cancer cells grow more rapidly than the blood vessels to nourish them; thus, as solid tumors grow, they are unable to obtain oxygen efficiently. In other words, they begin to experience hypoxia. Under these conditions, glycolysis leading to lactic acid fermentation becomes the primary source of ATP. Glycolysis is made more efficient in hypoxic tumors by the action of a transcription factor, hypoxia-inducible transcription factor (HIF-1). In the absence of oxygen, HIF-1 increases the expression of most glycolytic enzymes and the glucose transporters GLUT1 and GLUT3 (Table 16.5). In fact, glucose uptake correlates with tumor aggressiveness and a poor prognosis. These adaptations by the cancer cells enable the tumor to survive until vascularization can occur. HIF-1 also stimulates the growth of new tumors by increasing the expression of signal molecules, such as vascular endothelial growth factor (VEGF), that facilitate the growth of blood vessels (Figure 16.23). Without such vascularization, the tumor would cease to grow and either die or remain harmlessly small. Efforts are underway to develop drugs that inhibit vascularization of tumors.

Table 16.5. Proteins in glucose metabolism encoded by genes regulated by hypoxia-inducible factor.

Table 16.5

Proteins in glucose metabolism encoded by genes regulated by hypoxia-inducible factor.

Figure 16.23. Alteration of Gene Expression in Tumors Due to Hypoxia.

Figure 16.23

Alteration of Gene Expression in Tumors Due to Hypoxia. The hypoxic conditions inside a tumor mass lead to the activation of the hypoxia-inducible transcription factor (HIF-1), which induces metabolic adaptation (increase in glycolytic enzymes) and activates (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22395


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