Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

CHAPTER 14 GLYCOLYSIS, GLUCONEOGENESIS, AND THE PENTOSE PHOSPHATE PATHWAY starch or glycogen, a cell can stockpile large quantities of hexose units while maintaining a relatively low cytosolic osmolarity. When energy demands increase, glucose can be released from these intracellular storage polymers and used to produce ATP either aerobically or anaerobically. Glucose is not only an excellent fuel, but also a remarkably versatile precursor, capable of supplying a huge array of metabolic intermediates for biosynthetic reactions. A bacterium such as Escherichia coli can obtain from glucose the carbon skeletons for every amino acid, nucleotide, coenzyme, fatty acid, or other metabolic intermediate it needs for growth. A comprehensive study of the metabolic fates of glucose would encompass hundreds or thousands of transformations. In animals and vascular plants, glucose has four major fates: it may be (1) used in the synthesis of complex polysaccharides destined for the extracellular space; (2) stored in cells (as a polysaccharide or as sucrose); (3) oxidized to a three-carbon compound (pyruvate) via glycolysis to provide ATP and metabolic intermediates; or (4) oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose 5-phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes (Fig. 14-1). FIGURE 14-1 Major pathways of glucose utilization. Although not the only possible fates for glucose, these four pathways are the most significant in most cells. Organisms that do not have access to glucose from other sources must make it. Photosynthetic organisms make glucose by first reducing atmospheric CO2 to trioses, then converting the trioses to glucose. Nonphotosynthetic cells make glucose from simpler three- and four-carbon precursors by the process of gluconeogenesis, effectively reversing glycolysis in a pathway that uses many of the glycolytic enzymes. These principles are central to understanding glucose metabolism, but many apply to all metabolic pathways: Metabolites like glucose are oen activated with a high- energy group before their catabolism. Glycolysis is a nearly universal 10-step metabolic pathway for producing ATP by the oxidation of glucose. In this process, two molecules of ATP are invested to activate glucose, but the products of the pathway include four ATP, as well as NADH (a form of reducing power) and the triose pyruvate, which can be metabolized further in other pathways. Glucose and other hexoses and hexose phosphates obtained from stored polysaccharides or dietary carbohydrates feed into the glycolytic pathway. By using a common pathway for a number of starting materials, the cell economizes on the number of enzymes that must be synthesized and simplifies the regulation of the common pathway. Pyruvate formed under anaerobic conditions is reduced to lactate with electrons from NADH, recycling NADH to NAD+ and allowing continued glycolysis in the processes of lactate or alcohol fermentation. Manipulation of the fermentable material and the microorganisms present allows the synthesis of a variety of industrial products and foods. Gluconeogenesis is the synthesis of glucose from simpler precursors like pyruvate and lactate. Although it uses seven of the ten enzymes that also act in glycolysis, gluconeogenesis must bypass three of the most exergonic steps in glycolysis with energetically favorable reactions unique to gluconeogenesis. Glycolysis and gluconeogenesis are reciprocally regulated so that both processes don’t occur simultaneously in a futile cycle. Most regulatory mechanisms act on reactions that are unique to each pathway. The pentose phosphate pathway is an alternative pathway for glucose oxidation. It yields pentoses for nucleotide synthesis and reduced cofactors for biosynthesis of fatty acids, sterols, and many other compounds. 14.1 Glycolysis In glycolysis (from the Greek glykys, “sweet” or “sugar,” and lysis, “splitting”), a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of ATP and NADH. Glycolysis was the first metabolic pathway to be elucidated and is probably the best understood. From Eduard Buchner’s discovery in 1897 of fermentation in cell-free extracts of yeast until the elucidation of the whole pathway in yeast and in muscle in the 1930s, the reactions of glycolysis were a major focus of biochemical research. These discoveries showed that the reactions of life could be explained chemically, without reliance on a mystical life force. This philosophical shi led physiologist Jacques Loeb to observe in 1906, “The history of this problem is instructive, as it warns us against considering problems as beyond our reach because they have not yet found their solution.” The development of methods of enzyme purification, the discovery and recognition of the importance of coenzymes such as NAD, and the discovery of the pivotal metabolic role of ATP and other phosphorylated compounds all came out of studies of glycolysis. The glycolytic enzymes of many species have long since been purified and thoroughly studied. 1 Glycolysis is an almost universal central pathway of glucose catabolism, the pathway with the largest flux of carbon in most cells. The glycolytic breakdown of glucose is the sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal medulla, brain, and sperm, for example). Some plant tissues that are modified to store starch (such as potato tubers) and some aquatic plants (watercress, for example) derive most of their energy from glycolysis; many anaerobic microorganisms are entirely dependent on glycolysis. In the course of evolution, the chemistry of the reactions of glycolysis has been completely conserved. Genome sequencing and structural studies have shown that the glycolytic enzymes of vertebrates are closely similar in amino acid sequence and three- dimensional structure to their homologs in yeast and spinach. Although some archaea and parasitic microorganisms lack one or more of the enzymes of glycolysis, they retain the core of the pathway. The glycolytic pathway, of central importance in itself, is governed by thermodynamic principles and regulatory mechanisms that are common to all pathways of cell metabolism. It serves as a model of principles we will revisit throughout Part II of this book. An Overview: Glycolysis Has Two Phases Before examining each step of the pathway in some detail, we take a look at glycolysis as a whole. As all sugar derivatives in glycolysis are the D isomers, we will usually omit the D designation except when emphasizing stereochemistry. The breakdown of the six-carbon glucose into two molecules of the three-carbon pyruvate occurs in 10 steps, the first 5 of which constitute the preparatory phase (Fig. 14-2a). In these reactions, glucose is first phosphorylated at the hydroxyl group on C-6 (step ). The glucose 6-phosphate thus formed is converted to fructose 6-phosphate (step ), which is again phosphorylated, this time at C-1, to yield fructose 1,6-bisphosphate (step ). For both phosphorylations, ATP is the phosphoryl group donor. FIGURE 14-2 The two phases of glycolysis. For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP and two NADH per molecule of glucose converted to pyruvate. The numbered reaction steps correspond to the numbered headings in the text discussion. Keep in mind that each phosphoryl group, represented here as , has two negative charges (−PO2−3 ). Fructose 1,6-bisphosphate is split to yield two different three- carbon molecules, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (step ); this is the “lysis” step that gives the pathway its name. The dihydroxyacetone phosphate is isomerized to form a second molecule of glyceraldehyde 3- phosphate (step ), ending the first phase of glycolysis. Note that two molecules of ATP are invested before the cleavage of glucose into two three-carbon pieces; there will be a good return on this investment. To summarize: in the preparatory phase of glycolysis the energy of ATP is invested, raising the free-energy content of the intermediates, and the carbon chains of all the metabolized hexoses are converted to a common product, glyceraldehyde 3- phosphate. The energy gain comes in the payoff phase of glycolysis (Fig. 14- 2b). Each molecule of glyceraldehyde 3-phosphate is oxidized and phosphorylated by inorganic phosphate (not by ATP) to form 1,3- bisphosphoglycerate (step ). Energy is then released as the two molecules of 1,3-bisphosphoglycerate are converted to two molecules of pyruvate (steps through ). Much of this energy is conserved by the coupled phosphorylation of four molecules of ADP to ATP. The net yield is two molecules of ATP per molecule of glucose used, because two molecules of ATP were invested in the preparatory phase. Energy is also conserved in the payoff phase in the formation of two molecules of the electron carrier NADH per molecule of glucose. In the sequential reactions of glycolysis, three types of chemical transformations are particularly noteworthy: (1) degradation of the carbon skeleton of glucose to yield pyruvate; (2) phosphorylation of ADP to ATP by compounds with high phosphoryl group transfer potential, formed during glycolysis; and (3) transfer of a hydride ion to NAD +, forming NADH. The overall chemical logic of the pathway is described in Figure 14-3.

FIGURE 14-3 The chemical logic of the glycolytic pathway. In this simplified version of the pathway, each molecule is shown in a linear form, with carbon and hydrogen atoms not depicted, in order to highlight chemical transformations. Remember that glucose and fructose are present mostly in their cyclized forms in solution, although they are transiently present in linear form at the active sites of some of the enzymes in this pathway. The preparatory phase, steps to , converts the six-carbon glucose into two three-carbon units, each of them phosphorylated. Oxidation of the three-carbon units is initiated in the payoff phase, steps to . To produce pyruvate, the chemical steps must occur in the order shown. ATP and NADH Formation Coupled to Glycolysis During glycolysis some of the energy of the glucose molecule is conserved in ATP, while much remains in the product, pyruvate. The overall equation for glycolysis is (14-1) For each molecule of glucose degraded to pyruvate, two molecules of ATP are generated from ADP and Pi, and two molecules of NADH are produced by the reduction of NAD +. The reduction of NAD + (see Fig. 13-24) proceeds by the enzymatic transfer of a hydride ion (:H−) from the aldehyde group of glyceraldehyde 3-phosphate to the nicotinamide ring of NAD +, yielding the reduced coenzyme NADH. The other hydrogen atom of the substrate molecule is released to the solution as H+. We can now resolve the equation of glycolysis into two processes — the conversion of glucose to pyruvate, which is exergonic: G lucose+ 2NAD + → 2pyruvate+ 2NAD H + 2H+ ΔG ′°1 =−146 kJ /mol (14-2) and the formation of ATP from ADP and Pi, which is endergonic: G lucose+ 2NAD + + 2AD P + 2Pi→ 2pyruvate+ 2NAD H + 2H+ 2AD P + 2Pi→ 2AT P + 2H2O ΔG ′°2 = 2(30.5 kJ /mol)= 61.0 kJ /mol (14-3) The sum of Equations 14-2 and 14-3 gives the overall standard free-energy change of glycolysis, ΔG ′°Sum: ΔG ′°Sum = ΔG ′°1 + ΔG ′°2 =−146 kJ /mol+ 61.0 kJ /mol =−85 kJ /mol Under standard conditions, and under the (nonstandard) conditions that prevail in a cell, glycolysis is an essentially irreversible process, driven to completion by a large net decrease in free energy. Energy Remaining in Pyruvate Glycolysis releases only a small fraction of the total available energy of the glucose molecule. The two molecules of pyruvate formed by glycolysis still contain most of the chemical potential energy of glucose, energy that can be extracted by oxidative reactions in the citric acid cycle (Chapter 16) and oxidative phosphorylation (Chapter 19) — aerobic processes. Under anaerobic conditions, pyruvate can be reduced to lactate or ethanol (Section 14.3). The oxidation of pyruvate is an important catabolic process, but pyruvate has anabolic fates as well. It can, for example, provide the carbon skeleton for the synthesis of the amino acid alanine or for the synthesis of fatty acids. We return to these anabolic reactions of pyruvate in later chapters. Importance of Phosphorylated Intermediates Each of the nine glycolytic intermediates between glucose and pyruvate is phosphorylated (Fig. 14-2). The phosphoryl groups have three functions. 1. Because the plasma membrane lacks transporters for phosphorylated sugars, the phosphorylated glycolytic intermediates cannot leave the cell. Aer the initial phosphorylation, no further energy is necessary to retain phosphorylated intermediates in the cell, despite the large difference in their intracellular and extracellular concentrations. 2. Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. Energy made available with the transfer of phosphoryl groups from compounds with phosphoanhydride bonds (such as those in ATP) is partially conserved in the formation of phosphate esters such as glucose 6-phosphate. Compounds with higher group transfer potential than ATP, which are formed in glycolysis (1,3-bisphosphoglycerate and phosphoenolpyruvate), donate phosphoryl groups to ADP to form ATP. 3. Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions (Chapter 6). The phosphate groups of ADP, ATP, and the glycolytic intermediates form complexes with M g2+, and the substrate binding sites of many glycolytic enzymes are specific for these M g2+ complexes. Most glycolytic enzymes require M g2+ for activity. The Preparatory Phase of Glycolysis Requires ATP In the preparatory phase of glycolysis, two molecules of ATP are invested and the hexose chain is cleaved into two triose phosphates. The realization that phosphorylated hexoses were intermediates in glycolysis came slowly and serendipitously. In 1906, Arthur Harden and William Young tested their hypothesis that inhibitors of proteolytic enzymes would stabilize the glucose- fermenting enzymes in yeast extract. They added blood serum (known to contain inhibitors of proteolytic enzymes) to yeast extracts and observed the predicted stimulation of glucose metabolism. However, in a control experiment intended to show that boiling the serum destroyed the stimulatory activity, they discovered that boiled serum was just as effective at stimulating glycolysis! Careful examination and testing of the contents of the boiled serum revealed that inorganic phosphate was responsible for the stimulation. Harden and Young soon discovered that glucose added to their yeast extract was converted to a hexose bisphosphate (the “Harden-Young ester,” eventually identified as fructose 1,6-bisphosphate). This was the beginning of a long series of investigations on the role of organic esters and anhydrides of phosphate in biochemistry, which has led to our current understanding of the central role of phosphoryl group transfer in biology. Phosphorylation of Glucose In the first step of glycolysis (Fig. 14-2), glucose is activated for subsequent reactions by its phosphorylation at C-6 to yield glucose 6-phosphate, with ATP as the phosphoryl donor: This reaction, which is irreversible under intracellular conditions, is catalyzed by hexokinase. Recall that kinases are enzymes that catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor nucleophile (see Fig. 13-8c). Kinases are a subclass of transferases (see Table 6-3). The acceptor in the case of hexokinase is a hexose, normally glucose, although hexokinase also catalyzes the phosphorylation of other common hexoses, such as fructose and mannose, in some tissues. Hexokinase, like many other kinases, requires M g2+ for its activity, because the true substrate of the enzyme is not AT P4− but the M gAT P2− complex (see Fig. 13-12). M g2+ shields the negative charges of the phosphoryl groups in ATP, making the terminal phosphorus atom an easier target for nucleophilic attack by an — OH of glucose. Hexokinase undergoes a profound change in shape, an induced fit, when it binds glucose; two domains of the protein move about 8 Å closer to each other when ATP binds (see Fig. 6-30). This movement brings bound ATP closer to a molecule of glucose also bound to the enzyme and blocks the access of water (from the solvent), which might otherwise enter the active site and attack (hydrolyze) the phosphoanhydride bonds of ATP. Like the other nine enzymes of glycolysis, hexokinase is a soluble, cytosolic protein. Hexokinase is present in nearly all organisms. The human genome encodes four different hexokinases (I to IV), all of which catalyze the same reaction, but differ in kinetics, regulation, and location. Two or more enzymes that catalyze the same reaction but are encoded by different genes are called isozymes (see Box 14-3). One of the isozymes present in hepatocytes, hexokinase IV (also called glucokinase), differs from other forms of hexokinase in kinetic and regulatory properties, with important physiological consequences that are described in Section 14.5. Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate The enzyme phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6- phosphate, an aldose, to fructose 6-phosphate, a ketose: The mechanism for this reaction involves an enediol intermediate (Fig. 14-4). The reaction proceeds readily in either direction, as might be expected from the relatively small change in standard free energy. MECHANISM FIGURE 14-4 The phosphohexose isomerase reaction. The ring opening and closing reactions (steps and ) are catalyzed by an active-site His residue, by mechanisms omitted here for simplicity. The proton (light red) initially at C-2 is made more easily abstractable by electron withdrawal by the adjacent carbonyl and nearby hydroxyl groups. A er its transfer from C-2 to the active-site Glu residue (a weak acid), the proton is freely exchanged with the surrounding solution; that is, the proton abstracted from C-2 in step is not necessarily the same one that is added to C-1 in step . Phosphorylation of Fructose 6- Phosphate to Fructose 1,6-Bisphosphate In the second of the two priming reactions of glycolysis, phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate: KEY CONVENTION Compounds that contain two phosphate or phosphoryl groups attached at different positions in the molecule are named bisphosphates (or bisphospho compounds); for example, fructose 1,6-bisphosphate and 1,3-bisphosphoglycerate. Compounds with two phosphates linked together as a pyrophosphoryl group are named diphosphates; for example, adenosine diphosphate (ADP). Similar rules apply for the naming of trisphosphates (such as inositol 1,4,5-trisphosphate; see p. 425) and triphosphates (such as adenosine triphosphate, ATP). The enzyme that forms fructose 1,6-bisphosphate is called PFK-1 to distinguish it from a second enzyme (PFK-2) that catalyzes the formation of fructose 2,6-bisphosphate from fructose 6- phosphate in a separate pathway (the roles of PFK-2 and fructose 2,6-bisphosphate are discussed in Section 14.5). The PFK-1 reaction is essentially irreversible under cellular conditions, and it is the first “committed” step in the glycolytic pathway; glucose 6-phosphate and fructose 6-phosphate have other possible fates, but fructose 1,6-bisphosphate is targeted for glycolysis. Phosphofructokinase-1 is subject to complex allosteric regulation; its activity is increased whenever the cell’s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), accumulate. The enzyme is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. In some organisms, fructose 2,6-bisphosphate (not to be confused with the PFK-1 reaction product, fructose 1,6- bisphosphate) is a potent allosteric activator of PFK-1. Ribulose 5- phosphate, an intermediate in the pentose phosphate pathway discussed in Section 14.6, also activates phosphofructokinase indirectly. The multiple layers of regulation of this step in glycolysis are discussed in greater detail in Section 14.5. Some bacteria and protists and perhaps all plants have a different phosphofructokinase (PP-PFK-1) that uses pyrophosphate (PPi), not ATP, as the phosphoryl group donor in the synthesis of fructose 1,6-bisphosphate: Fructose6-phosphate+ PPi M g2+ −−−−−−→ fructose1,6-bisphosphate+ ΔG ′°=−2.9kJ / We will discuss this enzyme in Chapter 20. Cleavage of Fructose 1,6-Bisphosphate The enzyme fructose 1,6-bisphosphate aldolase, oen called simply aldolase, catalyzes a reverse aldol condensation (Fig. 14-5; see Fig. 13-4). Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, namely, glyceraldehyde 3-phosphate (an aldose) and dihydroxyacetone phosphate (a ketose):

MECHANISM FIGURE 14-5 The class I aldolase reaction. Note that cleavage between C-3 and C-4 depends on the presence of the carbonyl group at C-2, which is converted to an imine on the enzyme. A and B represent amino acid residues that serve as general acid (A) or base (B). There are two classes of aldolases. Class I aldolases, found in animals and plants, use the mechanism shown in Figure 14-5. Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2; the Zn2+ polarizes the carbonyl group and stabilizes the enolate intermediate created in the C— C bond cleavage step (see Fig. 6-23). Although the aldolase reaction has a strongly positive standard free-energy change in the direction of fructose 1,6-bisphosphate cleavage, at the lower concentrations of reactants present in cells the actual free-energy change is small and the aldolase reaction is readily reversible. We shall see later that aldolase acts in the reverse direction during the process of gluconeogenesis. Interconversion of the Triose Phosphates Only one of the two triose phosphates formed by aldolase, glyceraldehyde 3-phosphate, can be directly degraded in the subsequent steps of glycolysis. The other product, dihydroxyacetone phosphate, is immediately and reversibly converted to glyceraldehyde 3-phosphate by the fih enzyme of the glycolytic sequence, triose phosphate isomerase: The reaction mechanism is similar to the reaction promoted by phosphohexose isomerase in step of glycolysis (Fig. 14-4). Aer the triose phosphate isomerase reaction, the carbon atoms derived from C-1, C-2, and C-3 of the starting glucose are chemically indistinguishable from C-6, C-5, and C-4, respectively (Fig. 14-6); both “halves” of glucose have yielded glyceraldehyde 3-phosphate. FIGURE 14-6 Fate of the glucose carbons in the formation of glyceraldehyde 3-phosphate. (a) The origin of the carbons in the two three- carbon products of the aldolase and triose phosphate isomerase reactions. The end product of the two reactions is glyceraldehyde 3-phosphate (two molecules). (b) Each carbon of glyceraldehyde 3-phosphate is derived from either of two specific carbons of glucose. Note that the numbering of the carbon atoms of glyceraldehyde 3-phosphate differs from that of the glucose from which it is derived. In glyceraldehyde 3-phosphate, the most complex functional group (the carbonyl) is specified as C-1. This numbering change is important for interpreting experiments with glucose in which a single carbon is labeled with a radioisotope. (See Problems 5 and 22 at the end of this chapter.) This reaction completes the preparatory phase of glycolysis. The hexose molecule has been phosphorylated at C-1 and C-6 and then cleaved to form two molecules of glyceraldehyde 3- phosphate. The Payoff Phase of Glycolysis Yields ATP and NADH The payoff phase of glycolysis (Fig. 14-2b) includes the energy- conserving phosphorylation steps in which some of the chemical energy of the glucose molecule is conserved in the form of ATP and NADH. Remember that one molecule of glucose yields two molecules of glyceraldehyde 3-phosphate, and both halves of the glucose molecule follow the same pathway in the second phase of glycolysis. The conversion of two molecules of glyceraldehyde 3- phosphate to two molecules of pyruvate is accompanied by the formation of four molecules of ATP from ADP. However, the net yield of ATP per molecule of glucose degraded is only two, because two ATP were invested in the preparatory phase of glycolysis to phosphorylate the two ends of the hexose molecule. Oxidation of Glyceraldehyde 3- Phosphate to 1,3-Bisphosphoglycerate The first step in the payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3-phosphate dehydrogenase: This is the first of the two energy-conserving reactions of glycolysis that eventually lead to the formation of ATP. The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid. This type of anhydride, called an acyl phosphate, has a very high standard free energy of hydrolysis (ΔG′°=−49.3kJ /mol; see Table 13-6). Much of the free energy of oxidation of the aldehyde group of glyceraldehyde 3-phosphate is conserved by formation of the acyl phosphate group at C-1 of 1,3- bisphosphoglycerate. Glyceraldehyde 3-phosphate is covalently bound to the dehydrogenase during the reaction (Fig. 14-7). The aldehyde group of glyceraldehyde 3-phosphate reacts with the — SH group of an essential Cys residue in the active site, in a reaction analogous to the formation of a hemiacetal (see Fig. 7-5), in this case producing a thiohemiacetal. Reaction of the essential Cys residue with a heavy metal such as Hg2+ irreversibly inhibits the enzyme.

MECHANISM FIGURE 14-7 The glyceraldehyde 3-phosphate dehydrogenase reaction. The amount of NAD + in a cell (≤ 10−5M ) is far smaller than the amount of glucose metabolized in a few minutes. Glycolysis would soon come to a halt if the NADH formed in this step of glycolysis were not continuously reoxidized and recycled. We return to a discussion of this recycling of NAD + later in the chapter. Phosphoryl Transfer from 1,3- Bisphosphoglycerate to ADP The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3- bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate: Notice that phosphoglycerate kinase is named for the reverse reaction, in which it transfers a phosphoryl group from ATP to 3- phosphoglycerate. Like all enzymes, it catalyzes the reaction in both directions. This enzyme acts in the direction suggested by its name during gluconeogenesis (see Fig. 14-16) and during photosynthetic CO2 assimilation (see Fig. 20-26). In glycolysis, the reaction it catalyzes proceeds as shown above, in the direction of ATP synthesis. Steps and of glycolysis together constitute an energy- coupling process in which 1,3-bisphosphoglycerate is the common intermediate; it is formed in the first reaction (which would be endergonic in isolation), and its acyl phosphate group is transferred to ADP in the second reaction (which is strongly exergonic). The sum of these two reactions is G lyceraldehyde3-phosphate+ AD P + Pi+ NAD + ⇌ 3-phosphoglycerate+ AT P + NAD H + H+ΔG′°=−12.2kJ /mol Thus the overall reaction is exergonic. Recall from Chapter 13 that the actual free-energy change, ΔG, is determined by the standard free-energy change, ΔG′°, and the mass-action ratio, Q, which is the ratio [products]/[reactants] (see Eqn 13-4). For step , ΔG= ΔG′°+RTlnQ = ΔG′°+RTln [1,3-bisphosphoglycerate][NAD H] [glyceraldehyde3-phosphate][Pi][NAD +] Notice that [H+] is not included in Q. In biochemical calculations, [H+] is assumed to be a constant (10−7M ), and this constant is included in the definition of ΔG′° (p. 468). When the mass-action ratio is less than 1.0, its natural logarithm has a negative sign. In the cytosol, where these reactions are taking place, the ratio [NAD H]/[NAD +] is a small fraction, contributing to a low Q. Step , by consuming the product of step (1,3-bisphosphoglycerate), keeps [1,3-bisphosphoglycerate] relatively low in the steady state and thereby keeps Q for the overall energy-coupling process small. When Q is small, the contribution of ln Q can make ΔG strongly negative. This is simply another way of showing how the two reactions, steps and , are coupled through a common intermediate. The outcome of these coupled reactions, both reversible under cellular conditions, is that the energy released on oxidation of an aldehyde to a carboxylate group is conserved by the coupled formation of ATP from ADP and Pi. The formation of ATP by phosphoryl group transfer from a substrate such as 1,3- bisphosphoglycerate is referred to as a substrate-level phosphorylation, to distinguish this mechanism from respiration-linked phosphorylation. Substrate-level phosphorylations involve soluble enzymes and chemical intermediates (1,3-bisphosphoglycerate in this case). Respiration- linked phosphorylations, on the other hand, involve membrane- bound enzymes and transmembrane gradients of protons (Chapter 19). Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate The enzyme phosphoglycerate mutase catalyzes a reversible shi of the phosphoryl group between C-2 and C-3 of glycerate; M g2+ is essential for this reaction: The reaction occurs in two steps (Fig. 14-8). A phosphoryl group initially attached to a His residue of the mutase is transferred to the hydroxyl group at C-2 of 3-phosphoglycerate, forming 2,3- bisphosphoglycerate (2,3-BPG). The phosphoryl group at C-3 of 2,3-BPG is then transferred to the same His residue, producing 2- phosphoglycerate and regenerating the phosphorylated enzyme. Phosphoglycerate mutase is initially phosphorylated by phosphoryl transfer from 2,3-BPG, which is required in small quantities to initiate the catalytic cycle and is continuously regenerated by that cycle.

MECHANISM FIGURE 14-8 The phosphoglycerate mutase reaction. Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate In the second glycolytic reaction that generates a compound with high phosphoryl group transfer potential (the first was step ), enolase promotes reversible removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP): The mechanism of the enolase reaction involves an enolic intermediate stabilized by M g2+ (see Fig. 6-31). The reaction converts a compound with a relatively low phosphoryl group transfer potential (ΔG′° for hydrolysis of 2-phosphoglycerate is −17.6kJ /mol) to one with high phosphoryl group transfer potential (ΔG′° for PEP hydrolysis is −61.9kJ /mol) (see Fig. 13- 13). Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K+ and either M g2+ or M n2+:

In this substrate-level phosphorylation, the product pyruvate first appears in its enol form, then tautomerizes nonenzymatically to its keto form, which predominates at pH 7: The overall reaction has a large, negative standard free-energy change, due in large part to the spontaneous conversion of the enol form of pyruvate to the keto form (see Fig. 13-13). About half of the energy released by PEP hydrolysis (ΔG′°=−61.9kJ /mol) is conserved in the formation of the phosphoanhydride bond of ATP (ΔG′°=−30.5kJ /mol), and the rest (− 31.4kJ /mol) constitutes a large driving force pushing the reaction toward ATP synthesis. We discuss the regulation of pyruvate kinase in Section 14.5. The Overall Balance Sheet Shows a Net Gain of Two ATP and Two NADH Per Glucose We can now construct a balance sheet for glycolysis to account for (1) the fate of the carbon skeleton of glucose, (2) the input of Pi and ADP and output of ATP, and (3) the pathway of electrons in the oxidation-reduction reactions. The le side of the following equation shows all the inputs of ATP, NAD +, ADP, and Pi (consult Fig. 14-2), and the right side shows all the outputs (keep in mind that each molecule of glucose yields two molecules of pyruvate): G lucose+ 2AT P + 2NAD + + 4AD P + 2Pi→ 2pyruvate+ 2AD P + 2NAD H + 2H+ + 4AT P + 2H2O Canceling out common terms on both sides of the equation gives the overall equation for glycolysis: G lucose+ 2NAD + + 2AD P + 2Pi→ 2pyruvate+ 2NAD H + 2H+ + 2AT P + 2H2O In the overall glycolytic process, one molecule of glucose is converted to two molecules of pyruvate (the pathway of carbon). Two molecules of ADP and two of Pi are converted to two molecules of ATP (the pathway of phosphoryl groups). Four electrons, as two hydride ions, are transferred from two molecules of glyceraldehyde 3-phosphate to two of NAD + (the pathway of electrons). SUMMARY 14.1 Glycolysis Glycolysis is a near-universal pathway by which a glucose molecule is oxidized, in two phases, to two molecules of pyruvate, with energy conserved as ATP and NADH. Ten cytosolic enzymes act sequentially in glycolysis. The overall reaction converts glucose to two molecules of pyruvate, and energy is conserved in the synthesis of two molecules of ATP and two molecules of NADH. In the preparatory phase of glycolysis, two molecules of ATP are invested to activate glucose to fructose 1,6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate. In the payoff phase, each of the two molecules of glyceraldehyde 3-phosphate derived from glucose undergoes oxidation at C-1; some of the energy of this oxidation reaction is conserved in the form of one NADH and two ATP per triose phosphate oxidized. Subtracting the two ATP spent in the preparatory phase, the net equation for the overall process is G lucose+ 2NAD + + 2AD P + 2Pi→ 2 pyruvate+ 2NAD H + 2H+ + 2AT P + 2H2O 14.2 Feeder Pathways for Glycolysis Many carbohydrates besides glucose meet their catabolic fate in glycolysis, aer being transformed into one of the glycolytic intermediates. The most significant are the storage polysaccharides glycogen and starch, either within cells (endogenous) or obtained in the diet; the disaccharides maltose, lactose, and sucrose; and the monosaccharides fructose, mannose, and galactose (Fig. 14-9). FIGURE 14-9 Entry of dietary glycogen, starch, disaccharides, and hexoses into the preparatory stage of glycolysis. The numbered steps are described in the text. Endogenous Glycogen and Starch Are Degraded by Phosphorolysis Glycogen stored in animal tissues (primarily liver and skeletal muscle) and in microorganisms is mobilized for use within the same cell by a phosphorolytic reaction ( in Fig. 14-9) catalyzed by glycogen phosphorylase. The product of this reaction is not free glucose, but glucose 1-phosphate. We discuss glycogen metabolism in more detail in Chapter 15. In plant tissues, starch is mobilized by a similar phosphorolytic reaction catalyzed by starch phosphorylase. Glucose 1-phosphate produced by glycogen phos-phorylase is converted to glucose 6-phosphate by phosphoglucomutase , which catalyzes the reversible reaction G lucose1-phosphate⇌ glucose6-phosphate Phosphoglucomutase employs essentially the same mechanism as phosphoglycerate mutase (Fig. 14-8): both entail a bisphosphate intermediate, and the enzyme is transiently phosphorylated in each catalytic cycle. The general name mutase is given to enzymes that catalyze the transfer of a functional group from one position to another in the same molecule. Mutases are a subclass of isomerases, enzymes that interconvert stereoisomers or structural or positional isomers (see Table 6-3). The glucose 6-phosphate formed in the phosphoglucomutase reaction can continue through glycolysis or enter another pathway such as the pentose phosphate pathway, described in Section 14.6. WORKED EXAMPLE 14-1 Energy Savings for Glycogen Breakdown by Phosphorolysis Calculate the energy savings (in ATP molecules per glucose monomer) achieved by breaking down glycogen by phosphorolysis rather than hydrolysis to begin the process of glycolysis. SOLUTION: Phosphorolysis produces a phosphorylated glucose (glucose 1- phosphate), which is then converted to glucose 6-phosphate — without expenditure of the cellular energy (1 ATP) needed for formation of glucose 6-phosphate from free glucose. Thus only 1 ATP is consumed per glucose monomer in the preparatory phase, compared with 2 ATP when glycolysis starts with free glucose. The cell therefore gains 3 ATP per glucose monomer (4 ATP produced in the payoff phase minus 1 ATP used in the preparatory phase), rather than 2— a savings of 1 ATP per glucose monomer. Dietary Polysaccharides and Disaccharides Undergo Hydrolysis to Monosaccharides For most humans, starch is the major source of carbohydrates in the diet ( , Fig. 14-9). Dietary starch has essentially the same structure as glycogen, and its digestion proceeds by the same pathway. Digestion begins in the mouth, where salivary α - amylase hydrolyzes the internal (α1→ 4) glycosidic linkages of starch and glycogen, producing di- and trisaccharides. These are produced by hydrolysis reactions, in which water, not Pi, is the attacking species. In the stomach, salivary α -amylase is inactivated by the low pH, but a second form of α -amylase, secreted by the pancreas into the small intestine, continues the digestion process. Pancreatic α -amylase release into the small intestine yields mainly maltose and maltotriose (the di- and trisaccharides of glucose) and oligosaccharides called limit dextrins, fragments of amylopectin containing (α1→ 6) branch points, which are removed by limit dextrinases. Disaccharides are hydrolyzed by a family of membrane-bound hydrolases in the intestinal brush border: Only monosaccharides are taken up from the intestine. They are actively transported into the intestinal epithelial cells (see Fig. 11- 42), then passed into the blood to be carried to various tissues, where they are catabolized via glycolysis. As we noted in Chapter 7, most animals cannot digest cellulose for lack of the enzyme cellulase, which attacks the (β1→ 4) glycosidic bonds of cellulose. In ruminant animals, the extended stomach includes a chamber in which symbiotic microorganisms that produce cellulase break down cellulose into glucose molecules. These microorganisms use the resulting glucose in an anaerobic fermentation that produces large quantities of propionate. This propionate, aer conversion to succinate (see Fig. 17-12), serves as the starting material for gluconeogenesis, which produces much of the lactose in milk. Lactose Digestion and Lactose Intolerance The defining feature of mammals is, of course, mammary glands, which produce the disaccharide lactose for the nourishment of infants. The enzyme lactase converts lactose to glucose and galactose ( , Fig. 14-9), both of which are taken up from the small intestine and metabolized in the tissues by glycolysis. As infants are weaned, their lactase levels diminish, and lactase is absent in most adults — except in certain populations. About one in three adults in northern Europe and in some parts of Africa shows the lactase persistence phenotype. They continue to produce lactase and thus are able to digest milk into adulthood. The other two-thirds experience lactose intolerance due to the disappearance aer childhood of most or all of the lactase activity of the intestinal epithelial cells. Without intestinal lactase, lactose cannot be completely digested and absorbed in the small intestine, and it passes into the large intestine, where bacteria convert it to toxic products that cause abdominal cramps and diarrhea. The problem is further complicated because undigested lactose and its metabolites increase the osmolarity of the intestinal contents, favoring retention of water in the intestine, and causing diarrhea. In most parts of the world where lactose intolerance is prevalent, milk is not used as a food by adults, although milk products predigested with lactase are commercially available. In certain human disorders, several or all of the intestinal disaccharidases are missing. In these cases, the digestive disturbances triggered by dietary disaccharides can sometimes be minimized by a controlled diet lacking the undigestible carbohydrates. One way to determine whether lactase is present and active in the intestine is to compare the rise in blood glucose aer the ingestion of a quantity of either glucose or lactose. When glucose is ingested, the blood glucose level increases rapidly and transiently. When lactose is ingested, lactase, if present in the intestine, will hydrolyze the lactose into glucose and galactose and the blood glucose level will rise. If lactase is absent or less active, ingesting lactose will lead to little or no transient increase in blood glucose. Galactose Metabolism and Disease Galactose ( , Fig. 14-9), a product of the hydrolysis of lactose and therefore an important component in the diet of infants, passes in the blood from the intestine to the liver, where it is first phosphorylated at C-1, at the expense of ATP, by the enzyme galactokinase: G alactose+ AT P M g2+ −−−−−−→ galactose 1-phosphate AD P The galactose 1-phosphate is then transferred to a uridine nucleotide by a transferase. The resulting UDP-galactose is epimerized at C-4, forming UDP-glucose by a set of reactions in which UDP functions as an activator of hexose groups (Fig. 14-10) and a “tag” that these hexoses are in a separate pool from those destined for another process such as glycolysis. The epimerization, catalyzed by UDP-glucose 4-epimerase, involves first the oxidation of the C-4 — OH group to a ketone, then reduction of the ketone to an — OH, with inversion of the configuration at C-4. NAD is the cofactor for both the oxidation and the reduction. The glucose 1-phosphate made this way is converted to glucose 6-phosphate by phosphoglucomutase.

FIGURE 14-10 Conversion of galactose to glucose 1-phosphate. The conversion proceeds through a sugar-nucleotide derivative, UDP-galactose, which is formed when galactose 1-phosphate displaces glucose 1- phosphate from UDP-glucose. UDP-galactose is then converted by UDP- glucose 4-epimerase to UDP-glucose, in a reaction that involves oxidation of C-4 (light red) by NAD +, then reduction of C-4 by NADH; the result is inversion of the configuration at C-4. The UDP-glucose is recycled through another round of the same reaction. The net effect of this cycle is the conversion of galactose 1-phosphate to glucose 1-phosphate; there is no net production or consumption of UDP-galactose or UDP-glucose. Defects in the enzymes that catalyze each of these steps result in the various galactosemias shown. A defect in any of the enzymes in this pathway has serious medical consequences. In galactokinase-deficiency galactosemia, caused by a defect in the GALK gene, high galactose concentrations are found in blood and urine. Affected individuals develop cataracts in infancy, caused by deposition of the galactose metabolite galactitol in the lens. The other symptoms in this disorder are relatively mild, and strict limitation of galactose in the diet greatly diminishes their severity. Transferase-deficiency galactosemia, caused by a defect in the GALT gene, is more serious; it is characterized by poor growth in childhood, speech abnormality, mental deficiency, and liver damage that may be fatal, even when galactose is withheld from the diet. Epimerase-deficiency galactosemia, caused by a defect in the GALE gene, leads to similar symptoms, but they are less severe when dietary galactose is carefully controlled.

Fructose and Mannose In most organisms, hexoses other than glucose can undergo glycolysis aer conversion to a phosphorylated derivative. Fructose, present in free form in many fruits and formed by hydrolysis of sucrose in the small intestine of vertebrates, is phosphorylated by hexokinase: Fructose+ AT P M g2+ −−−−−−→ fructose6-phosphate+ AD P This is a major pathway of fructose entry into glycolysis in the muscles and kidney ( , Fig. 14-9). In the liver, fructose enters by a different pathway. The liver enzyme fructokinase catalyzes the phosphorylation of fructose at C-1 rather than C-6 ( , Fig. 14-9): Fructose+ AT P M g2+ −−−−−−→ fructose1-phosphate+ AD P The fructose 1-phosphate is then cleaved to glyceraldehyde and dihydroxyacetone phosphate by fructose 1-phosphate aldolase: Dihydroxyacetone phosphate is converted to glyceraldehyde 3- phosphate by the glycolytic enzyme triose phosphate isomerase. Glyceraldehyde is phosphorylated by ATP and triose kinase to glyceraldehyde 3-phosphate: Thus, both products of fructose 1-phosphate hydrolysis enter the glycolytic pathway as glyceraldehyde 3-phosphate. Mannose, released in the digestion of various polysaccharides and glycoproteins of foods, is phosphorylated at C-6 by hexokinase ( , Fig. 14-9): G lyceraldehyde+ AT P M g2+ −−−−−−→ glyceraldehyde3-phosphate+ AD M annose+ AT P M g2+ −−−−−−→ mannose6-phosphate+ AD P Phosphohexose isomerase converts mannose 6-phosphate to fructose 6-phosphate, which enters glycolysis. SUMMARY 14.2 Feeder Pathways for Glycolysis Endogenous glycogen and starch, polymeric storage forms of glucose, undergo sequential phosphorolysis of glucose residues, forming glucose 1-phosphate. Phosphoglucomutase converts the glucose 1-phosphate to glucose 6-phosphate, which can enter glycolysis at a point in the preparatory phase that requires the investment of only one more ATP. Ingested polysaccharides and disaccharides are converted to monosaccharides by hydrolytic enzymes in saliva and in the small intestine. The monosaccharides pass through intestinal cells to the bloodstream, which transports them to the liver or other tissues. Lactase is present in infants but oen absent in adults, producing lactose intolerance. D-Hexoses, including galactose, fructose, and mannose, can be phosphorylated and funneled into glycolysis. Galactose is converted to glucose 1-phosphate through UDP-galactose and UDP-glucose intermediates. A genetic defect in enzymes of this pathway results in one of several galactosemias of varying severity. 14.3 Fates of Pyruvate With the exception of some interesting variations in the bacterial realm, the pyruvate formed by glycolysis is further metabolized via one of three catabolic routes (Fig. 14-11). Under aerobic conditions, glycolysis is only the first stage in the complete degradation of glucose. The pyruvate formed in the final step of glycolysis is oxidized to acetate (acetyl-CoA), which enters the citric acid cycle and is oxidized completely to CO2 and H2O (Chapter 16). The electrons from these oxidations are carried by NADH and FAD H2, which are ultimately reoxidized to NAD + and FAD by passage of electrons to O2 through a chain of carriers in mitochondrial respiration, to form H2O. The energy from the electron-transfer reactions drives the synthesis of ATP (Chapter 19). FIGURE 14-11 Three possible catabolic fates of the pyruvate formed in glycolysis and the recycling of NADH. Red arrows follow the regeneration of NAD + from NADH. Under aerobic conditions, pyruvate is activated to acetyl-CoA and is completely oxidized to CO2 and water through the citric acid cycle and mitochondrial oxidative phosphorylation. NADH produced in this pathway is oxidized to NAD + through mitochondrial electron transfer. Under anaerobic conditions, pyruvate reduction to lactate or to ethanol is required to produce the NAD + needed for glycolysis to continue. Pyruvate also serves as a precursor in many anabolic reactions, not shown here. The earliest cells lived in an atmosphere almost devoid of oxygen and evolved deriving energy from fuel molecules under anaerobic conditions. Under anaerobic or low-oxygen conditions (hypoxia), NADH cannot be reoxidized to NAD + by passing its electrons to O2. But for glycolysis to continue, NAD + must be regenerated. Under these conditions, glucose is degraded by fermentation (defined below), which leads to one of two different fates for the pyruvate formed by glycolysis. In lactic acid fermentation, pyruvate accepts electrons from NADH and is reduced to lactate while regenerating the NAD + necessary for glycolysis. In ethanol (alcohol) fermentation, pyruvate is further catabolized to ethanol (Fig. 14-11). The Pasteur and Warburg Effects Are Due to Dependence on Glycolysis Alone for ATP Production During his studies on the fermentation of glucose by yeast, Louis Pasteur discovered that both the rate and the total amount of glucose consumption under anaerobic conditions were many times greater than under aerobic conditions. Later studies of muscle showed the same large difference in the rates of glycolysis under anaerobic and aerobic conditions. The biochemical basis of this “Pasteur effect” is now clear. The ATP yield from glycolysis alone (2 ATP per molecule of glucose) is much smaller than that from the complete oxidation of glucose to CO2 under aerobic conditions (30 or 32 ATP per glucose; see Table 19-5). About 15 times as much glucose must therefore be consumed anaerobically as aerobically to yield the same amount of ATP. The flux of glucose through the glycolytic pathway is regulated to maintain nearly constant ATP levels (as well as adequate supplies of glycolytic intermediates that serve biosynthetic roles). The required adjustment in the rate of glycolysis is achieved by a complex interplay among ATP consumption, NAD + regeneration from NADH formed in glycolysis, and allosteric regulation of several glycolytic enzymes — including hexokinase, PFK-1, and pyruvate kinase — and by second-to-second fluctuations in the concentration of key metabolites that reflect the cellular balance between ATP production and consumption. On a slightly longer time scale, glycolysis is regulated by the hormones glucagon, epinephrine, and insulin, and by changes in the expression of the genes for several glycolytic enzymes. An especially interesting case is glycolysis in tumors. The German biochemist Otto Warburg first observed in 1928 that tumors of nearly all types carry out glycolysis at a much higher rate than normal tissue, even when oxygen is available. This “Warburg effect” is the basis for several methods of detecting and treating cancer (Box 14-1). BOX 14-1 MEDICINE High Rate of Glycolysis in Tumors Suggests Targets for Chemotherapy and Facilitates Diagnosis In many types of tumors found in humans and other animals, glucose uptake and glycolysis proceed about 10 times faster than in normal, noncancerous tissues. Most tumor cells grow under hypoxic conditions (i.e., with limited oxygen supply) because, at least initially, they lack the capillary network to supply sufficient oxygen. Cancer cells located more than 100to200μM from the nearest capillaries must depend on glycolysis alone (without further oxidation of pyruvate) for much of their ATP production. The energy yield (2 ATP per glucose) is far lower than can be obtained by the complete oxidation of pyruvate to CO2 in mitochondria (about 30 ATP per glucose; Chapter 19). So, to make the same amount of ATP, tumor cells must take up much more glucose than do normal cells, converting it to pyruvate and then to lactate as they recycle NADH. It is likely that two early steps in the transformation of a normal cell into a tumor cell are (1) the change to dependence on glycolysis alone for ATP production and (2) the development of tolerance to a low pH in the extracellular fluid (caused by release of lactic acid). In general, the more aggressive the tumor, the greater its rate of glycolysis. This increase in glycolysis is achieved, at least in part, by increased synthesis of the glycolytic enzymes and of the plasma membrane transporters GLUT1 and GLUT3 (see Table 11-1) that carry glucose into cells. (GLUT1 and GLUT3 are not dependent on insulin.) The hypoxia-inducible transcription factor (HIF-1) is a protein that acts at the level of mRNA synthesis to stimulate the production of at least eight glycolytic enzymes and the glucose transporters when oxygen supply is limited (Fig. 1). With the resulting high rate of glycolysis, the tumor cell can survive anaerobic conditions until the supply of blood vessels has caught up with tumor growth. Another protein induced by HIF-1 is the peptide hormone VEGF (vascular endothelial growth factor), which stimulates the outgrowth of blood vessels (angiogenesis) toward the tumor. FIGURE 1 The anaerobic metabolism of glucose in tumor cells yields far less ATP (2 per glucose) than the complete oxidation to CO2 that takes place in healthy cells under aerobic conditions (~30 ATP per glucose), so a tumor cell must consume much more glucose to produce the same amount of ATP. Glucose transporters and most of the glycolytic enzymes are overproduced in tumors. Compounds that inhibit hexokinase, glucose 6-phosphate dehydrogenase, or transketolase block ATP production by glycolysis, thus depriving the cancer cell of energy and killing it. There is also evidence that the tumor suppressor protein p53, which is mutated in most types of cancer (see Section 12.9), controls the synthesis and assembly of mitochondrial proteins essential to the passage of electrons to O2. Cells with mutant p53 are defective in mitochondrial electron transfer and are forced to rely more heavily on glycolysis for ATP production (Fig. 1). This heavier reliance of tumors than of normal tissue on glycolysis suggests a possibility for anticancer therapy: inhibitors of glycolysis might target and kill tumors by depleting their supply of ATP. Three inhibitors of hexokinase have shown promise as chemotherapeutic agents: 2-deoxyglucose, lonidamine, and 3-bromopyruvate. By preventing the formation of glucose 6-phosphate, these compounds not only deprive tumor cells of glycolytically produced ATP but also prevent the formation of pentose phosphates via the pentose phosphate pathway, which also begins with glucose 6-phosphate. Without pentose phosphates, a cell cannot synthesize the nucleotides essential to DNA and RNA synthesis and thus cannot grow or divide. Another anticancer drug already approved for clinical use is imatinib (Gleevec), described in Box 12-4. It inhibits a specific tyrosine kinase, preventing the increased synthesis of hexokinase normally triggered by that kinase. The thiamine analog oxythiamine, which blocks the action of a transketolase-like enzyme that converts xylulose 5- phosphate to glyceraldehyde 3-phosphate (Fig. 1), is in preclinical trials as an antitumor drug. The high glycolytic rate in tumor cells also has diagnostic usefulness. The relative rates at which tissues take up glucose can be used in some cases to pinpoint the location of tumors. In positron emission tomography (PET), individuals are injected with a harmless, isotopically labeled glucose analog that is taken up but not metabolized by tissues. The labeled compound is 2- fluoro-2-deoxyglucose (FdG), in which the hydroxyl group at the C-2 of glucose is replaced with 18F (Fig. 2). This compound is taken up via GLUT transporters and is a good substrate for hexokinase, but it cannot be converted to the enediol intermediate in the phosphohexose isomerase reaction (see Fig. 14-4) and therefore accumulates as 6-phospho-FdG. The extent of its accumulation depends on its rate of uptake and phosphorylation, which, as noted above, is typically 10 or more times higher in tumors than in normal tissue. Decay of 18F yields positrons (two per 18F atom) that can be detected by a series of sensitive detectors positioned around the body, which allows accurate localization of accumulated 6-phospho-FdG (Fig. 3). FIGURE 2 Phosphorylation of 18F-labeled 2-fluoro-2-deoxyglucose by hexokinase traps the FdG in cells (as 6-phospho-FdG), where its presence can be detected by positron emission from 18F. FIGURE 3 Detection of cancerous tissue by positron emission tomography (PET). The adult male patient had undergone surgical removal of a primary skin cancer (malignant melanoma). The image on the le , obtained by whole-body computed tomography (CT scan), shows the location of the so tissues and bones. The central panel is a PET scan a er the patient had ingested 18F-labeled 2-fluoro-2- deoxyglucose (FdG). Dark spots indicate regions of high glucose utilization. As expected, the brain and bladder are heavily labeled — the brain because it uses most of the glucose consumed in the body, and the bladder because the 18F- labeled 6-phospho-FdG is excreted in the urine. When the intensity of the label in the PET scan is translated into false color (the intensity increases from green to yellow to red) and the image is superimposed on the CT scan, the fused image (right) reveals cancer in the bones of the upper spine, in the liver, and in some regions of muscle, all the result of cancer spreading from the primary malignant melanoma. Otto Warburg, 1883–1970 Warburg is generally considered the preeminent biochemist of the first half of the twentieth century. He made seminal contributions to many other areas of biochemistry, including respiration, photosynthesis, and the enzymology of intermediary metabolism. Beginning in 1930, Warburg and his associates purified and crystallized seven of the enzymes of glycolysis. They developed an experimental tool that revolutionized biochemical studies of oxidative metabolism: the Warburg manometer, which directly measured the oxygen consumption of tissues by monitoring changes in gas volume, and thus allowed quantitative measurement of any enzyme with oxidase activity. Pyruvate Is the Terminal Electron Acceptor in Lactic Acid Fermentation When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD + is regenerated from NADH by the reduction of pyruvate to lactate. Some tissues and cell types (such as erythrocytes, which have no mitochondria and thus cannot oxidize pyruvate to CO2) produce lactate from glucose even under aerobic conditions. The reduction of pyruvate in this pathway is catalyzed by lactate dehydrogenase, which forms the L isomer of lactate at pH 7: The overall equilibrium of the reaction strongly favors lactate formation, as shown by the large negative standard free-energy change. In glycolysis, dehydrogenation of the two molecules of glyceraldehyde 3-phosphate derived from each molecule of glucose converts two molecules of NAD + to two of NADH. Because the reduction of two molecules of pyruvate to two of lactate regenerates two molecules of NAD +, there is no net change in NAD + or NADH: The lactate formed by active skeletal muscles (or by erythrocytes or retinal cells) can be recycled; it is carried in the blood to the liver, where it is converted to glucose during the recovery from strenuous muscular activity. When lactate is produced in large quantities during vigorous muscle contraction (during a sprint, for example), the acidification that results from ionization of lactic acid in muscle and blood limits the period of vigorous activity. The best-conditioned athletes can sprint at top speed for no more than a minute (Box 14-2). BOX 14-2 Glucose Catabolism at Limiting Concentrations of Oxygen Most vertebrates are essentially aerobic organisms; they convert glucose to pyruvate by glycolysis, then use molecular oxygen to oxidize the pyruvate completely to CO2 and H2O. Anaerobic catabolism of glucose to lactate occurs during short bursts of extreme muscular activity — for example, in a sprint — during which oxygen cannot be carried to the muscles fast enough to oxidize pyruvate. Instead, the muscles use their stored glucose (glycogen) as fuel to generate ATP by fermentation, with lactate as the end product. In a sprint, lactate in the blood builds up to high concentrations. It is slowly converted back to glucose by gluconeogenesis in the liver in the subsequent rest or recovery period, during which oxygen is consumed at a gradually diminishing rate until the breathing rate returns to normal. The excess oxygen consumed in the recovery period represents a repayment of the oxygen debt. This is the amount of oxygen required to supply ATP for gluconeogenesis during recovery respiration, in order to regenerate the glycogen “borrowed” from liver and muscle to carry out intense muscular activity in the sprint. The cycle of reactions that includes glucose conversion to lactate in muscle and lactate conversion to glucose in liver is called the Cori cycle, for Carl and Gerty Cori, whose studies in the 1930s and 1940s clarified the pathway and its role (see Box 15-1). The circulatory systems of most small vertebrates can carry oxygen to their muscles fast enough to avoid having to use muscle glycogen anaerobically. For example, migrating birds o en fly great distances at high speeds without rest and without incurring an oxygen debt. Many running animals of moderate size also maintain an essentially aerobic metabolism in their skeletal muscle. However, the circulatory systems of larger animals, including humans, cannot completely sustain aerobic metabolism in skeletal muscles over long periods of intense muscular activity. These animals generally are slow-moving under normal circumstances and engage in intense muscular activity only in the gravest emergencies, because such bursts of activity require long recovery periods to repay the oxygen debt. Alligators and crocodiles, for example, are normally sluggish animals. Yet, when provoked, they are capable of lightning-fast charges and dangerous lashings of their powerful tails. Such intense bursts of activity are short and must be followed by long periods of recovery. The fast emergency movements require lactic acid fermentation to generate ATP in skeletal muscles. The stores of muscle glycogen are rapidly expended in intense muscular activity, and lactate reaches very high concentrations in myocytes and extracellular fluid. Whereas a trained athlete can recover from a 100 m sprint in 30 min or less, an alligator may require many hours of rest and extra oxygen consumption to clear the excess lactate from its blood and regenerate muscle glycogen a er a burst of activity. Other large animals, such as the elephant and the rhinoceros, have similar metabolic characteristics, as do diving mammals such as whales and seals. Dinosaurs and other huge, now-extinct animals probably had to depend on lactic acid fermentation to supply energy for muscular activity, followed by very long recovery periods during which they were vulnerable to attack by smaller predators that were better able to use oxygen and thus better adapted to continuous, sustained muscular activity. Deep-sea explorations have revealed many species of marine life at great ocean depths, where the oxygen concentration is near zero. For example, the primitive coelacanth, a large fish recovered from depths of 4,000 m or more off the coast of South Africa, has an essentially anaerobic metabolism in virtually all its tissues. It converts carbohydrates to lactate and other products, most of which must be excreted. Some marine vertebrates ferment glucose to ethanol and CO2 in order to generate ATP. Francena McCorory, Olympic sprinter Although conversion of glucose to lactate includes two oxidation- reduction steps, there is no net change in the oxidation state of carbon; in glucose (C6H12O6) and lactic acid (C3H6O3), the H:C ratio is the same. Nevertheless, some of the energy of the glucose molecule has been extracted by its conversion to lactate — enough to give a net yield of two molecules of ATP for every glucose molecule consumed. Fermentation is the general term for such processes, which extract energy (as ATP) but do not consume oxygen or change the concentrations of NAD + or NADH. Ethanol Is the Reduced Product in Ethanol Fermentation Yeast and other microorganisms ferment glucose to ethanol and CO2, rather than to lactate. Glucose is metabolized to pyruvate by glycolysis, and the pyruvate is converted to ethanol and CO2 in a two-step process: In the first step, pyruvate is decarboxylated to form acetaldehyde in an irreversible reaction catalyzed by pyruvate decarboxylase. This reaction is a simple decarboxylation and does not involve the net oxidation of pyruvate. Pyruvate decarboxylase requires M g2+ and has a tightly bound coenzyme, thiamine pyrophosphate, which is discussed below. In the second step, acetaldehyde is reduced to ethanol through the action of alcohol dehydrogenase, with the reducing power furnished by NADH derived from the dehydrogenation of glyceraldehyde 3-phosphate. This reaction is a well-studied case of hydride transfer from NADH (Fig. 14-12). Ethanol and CO2 are thus the end products of ethanol fermentation, and the overall equation is Glucose+ 2AD P + 2Pi→ 2ethanol+ 2CO2+ 2AT P + 2H2O As in lactic acid fermentation, there is no net change in the ratio of hydrogen to carbon atoms when glucose (H:Cratio= 12/6= 2) is fermented to two ethanol and two CO2 (combined H:Cratio= 12/6= 2). In all fermentations, the H:C ratio of the reactants and products remains the same. MECHANISM FIGURE 14-12 The alcohol dehydrogenase reaction. Pyruvate decarboxylase is present in brewer’s and baker’s yeast (different strains of the species Saccharomyces cerevisiae) and in all other organisms that ferment glucose to ethanol, including some plants. The CO2 produced by pyruvate decarboxylation in brewer’s yeast is responsible for the characteristic carbonation of champagne. The ancient art of brewing beer involves several enzymatic processes in addition to the reactions of ethanol fermentation. In baking, CO2 released by pyruvate decarboxylase when yeast is mixed with a fermentable sugar causes dough to rise. The enzyme is absent in vertebrate tissues and in other organisms that carry out lactic acid fermentation. Alcohol dehydrogenase is present in many organisms that metabolize ethanol, including humans. In the liver it catalyzes the oxidation of ethanol, either ingested or produced by intestinal microorganisms, with the concomitant reduction of NAD + to NADH. In this case, the reaction proceeds in the direction opposite to that involved in the production of ethanol by fermentation. The pyruvate decarboxylase reaction provides our first encounter with thiamine pyrophosphate (TPP) (Fig. 14-13), a coenzyme derived from vitamin B1. TPP plays an important role in the cleavage of bonds adjacent to a carbonyl group, such as the decarboxylation of α -keto acids, and in chemical rearrangements in which an activated acetaldehyde group is transferred from one carbon atom to another (Table 14-1). The functional part of TPP, the thiazolium ring, has a relatively acidic proton at C-2. Loss of this proton produces a carbanion that is the active species in TPP- dependent reactions. The carbanion readily adds to carbonyl groups, and the thiazolium ring is thereby positioned to act as an “electron sink” that greatly facilitates reactions such as the decarboxylation catalyzed by pyruvate decarboxylase.

MECHANISM FIGURE 14-13 Thiamine pyrophosphate (TPP) and its role in pyruvate decarboxylation. (a) TPP is the coenzyme form of vitamin B1 (thiamine). The reactive carbon atom in the thiazolium ring of TPP is shown in red. In the reaction catalyzed by pyruvate decarboxylase, two of the three carbons of pyruvate are carried transiently on TPP in the form of a hydroxyethyl, or “active acetaldehyde,” group (b), which is subsequently released as acetaldehyde. (c) The thiazolium ring of TPP stabilizes carbanion intermediates by providing an electrophilic (electron-deficient) structure into which the carbanion electrons can be delocalized by resonance. Structures with this property, o en called “electron sinks,” play a role in many biochemical reactions — here, facilitating carbon–carbon bond cleavage. Dietary insufficiency of thiamine causes the serious disease beriberi and the Wernicke-Korsakoff syndrome. TABLE 14-1 Some TPP-Dependent Reactions Enzyme Pathway(s) Bond cleaved Bond formed Pyruvate decarboxylase Ethanol fermentation Pyruvate dehydrogenase α -Ketoglutarate dehydrogenase Synthesis of acetyl-CoA Citric acid cycle Transketolase Carbon- assimilation reactions Pentose phosphate pathway Fermentations Produce Some Common Foods and Industrial Chemicals Our progenitors learned millennia ago to use fermentation in the production and preservation of foods and beverages. Certain microorganisms present in raw food products ferment the carbohydrates and yield metabolic products that give the foods their characteristic forms, textures, and tastes. In modern times, industrial fermentation produces organic chemicals and fuels. Fermented Foods Yogurt, already known in biblical times, is produced when the bacterium Lactobacillus bulgaricus ferments the carbohydrate in milk, producing lactic acid; the resulting drop in pH causes the milk proteins to precipitate, producing the thick texture and sour taste of unsweetened yogurt. Another bacterium, Propionibacterium freudenreichii, ferments milk to produce propionic acid and CO2; the propionic acid precipitates milk proteins, and bubbles of CO2 cause the holes characteristic of Swiss cheese. Many other food products are the result of fermentations: pickles, sauerkraut, sausage, soy sauce, kimchi, kefir, dahi, and kombucha. The drop in pH associated with fermentation also helps to preserve foods, because most of the microorganisms that cause food spoilage cannot grow at low pH. In agriculture, plant byproducts such as corn stalks are preserved for use as animal feed by packing them into a large container (a silo) with limited access to air; microbial fermentation produces acids that lower the pH. The silage that results from this fermentation process can be kept as animal feed for long periods without spoilage. Fermented Beverages Beer brewing was a science learned early in human history, and later refined for larger-scale production (Fig. 14-14). Brewers prepare beer by ethanol fermentation of the carbohydrates in cereal grains (seeds) such as barley, carried out by yeast glycolytic enzymes. The carbohydrates, largely polysaccharides, must first be degraded to disaccharides and monosaccharides. In the malting process, the barley seeds are allowed to germinate until they form the hydrolytic enzymes required to break down their polysaccharides, at which point germination is stopped by controlled heating. The product is malt, which contains enzymes that catalyze the hydrolysis of the β linkages of cellulose and other cell wall polysaccharides of the barley husks, and enzymes such as α -amylase and maltase. The malt is mixed with water, mashed, and boiled with hops to add flavor. Yeast cells added to this mixture grow and reproduce rapidly, using energy obtained from available sugars. No ethanol forms during this stage, because the yeast, amply supplied with oxygen, oxidizes the pyruvate formed by glycolysis to CO2 and H2O via the citric acid cycle. FIGURE 14-14 Beer brewing. Large breweries and microbreweries produce beers with a wide variety of flavors, the result of differences in materials and fermentation conditions. When all the dissolved oxygen in the vat has been consumed, the yeast cells switch to anaerobic metabolism and ferment the sugars into ethanol and CO2. The fermentation process is controlled in part by the concentration of the ethanol formed, by the pH, and by the amount of remaining sugar. Aer fermentation has been stopped, the cells are removed and the “raw” beer is ready for final processing. Chemical Production by Fermentation In 1910, Chaim Weizmann (later to become the first president of Israel) discovered that the bacterium Clostridium acetobutyricum ferments starch to butanol and acetone. This discovery opened the field of industrial fermentations, in which some readily available material rich in carbohydrate is supplied to a pure culture of a specific microorganism, which ferments it into a product of greater commercial value. Microbial fermentations produce formic, acetic, propionic, butyric, and succinic acids, and ethanol, glycerol, methanol, isopropanol, butanol, and butanediol. Industrial fermentations are also used to produce certain antibiotics, including penicillin, streptomycin, and chloramphenicol. These fermentations are generally carried out in huge closed vats in which temperature and access to air are controlled to favor the multiplication of the desired microorganism and to exclude contaminating organisms. The beauty of industrial fermentations is that complicated, multistep chemical transformations are carried out in high yields and with few side products by chemical factories that reproduce themselves — microbial cells. Fuel Production by Fermentation Much of the technology developed for large-scale production of alcoholic beverages can be applied to the production of ethanol as a renewable fuel. The principal advantage of ethanol as a fuel is that it can be produced from relatively inexpensive and renewable resources rich in sucrose, starch, or cellulose — starch from corn or wheat; sucrose from beets or cane; and cellulose from straw, forest industry waste, or municipal solid waste. Typically, the raw material (feedstock) is converted chemically to monosaccharides, then fed to a hardy strain of yeast in an industrial-scale fermenter. The fermentation can yield not only ethanol for fuel but also side products such as proteins that can be used as animal feed. SUMMARY 14.3 Fates of Pyruvate The NADH formed in glycolysis must be recycled to regenerate NAD +, which is required as an electron acceptor in the first step of the payoff phase. Under aerobic conditions, electrons pass from NADH to O2 in mitochondrial respiration. The Warburg effect is the observation that tumor cells have high rates of glycolysis, with fermentation of glucose to lactate, even in the presence of oxygen. It is the basis of PET scanning used to diagnose tumors. Under anaerobic or hypoxic conditions, many organisms regenerate NAD + by transferring electrons from NADH to pyruvate, forming lactate. Other organisms, such as yeast, regenerate NAD + by reducing pyruvate to ethanol and CO2. A variety of microorganisms can ferment sugar in fresh foods, resulting in changes in pH, taste, and texture, and preserving food from spoilage. Fermentations are used in industry to produce many commercially valuable organic compounds from inexpensive starting materials. 14.4 Gluconeogenesis The central role of glucose in metabolism arose early in evolution, and this sugar remains the nearly universal fuel and building block in modern organisms, from microbes to humans. In mammals, some tissues depend almost completely on glucose for their metabolic energy. For the human brain and nervous system, as well as the erythrocytes, testes, renal medulla, and embryonic tissues, glucose from the blood is the sole or major fuel source. The brain alone requires about 120 g of glucose each day — more than half of all the glucose stored as glycogen in muscle and liver. However, the supply of glucose from these stores is not always sufficient; between meals and during longer fasts, or aer vigorous exercise, glycogen is depleted. For these times, organisms need a method for synthesizing glucose from noncarbohydrate precursors. This is accomplished by a pathway called gluconeogenesis (“new formation of sugar”), which converts pyruvate and related three- and four-carbon compounds to glucose. Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms. The reactions are essentially the same in all tissues and all species. The important precursors of glucose in animals are three-carbon compounds such as lactate, pyruvate, and glycerol, as well as certain amino acids (Fig. 14-15). In mammals, gluconeogenesis takes place mainly in the liver, and to a lesser extent in the renal cortex and in the epithelial cells that line the small intestine. The glucose produced passes into the blood to supply other tissues. Aer vigorous exercise, lactate produced by anaerobic glycolysis in skeletal muscle returns to the liver and is converted to glucose, which moves back to muscle and is converted to glycogen — a circuit called the Cori cycle (see Fig. 23-17). In plant seedlings, stored fats and proteins are converted, via paths that include gluconeogenesis, to the disaccharide sucrose for transport throughout the developing plant. Glucose and its derivatives are precursors for the synthesis of plant cell walls, nucleotides and coenzymes, and a variety of other essential metabolites. In many microorganisms, gluconeogenesis starts from simple organic compounds of two or three carbons, such as acetate, lactate, and propionate, in their growth medium. FIGURE 14-15 Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle, which we discuss in Chapter 16. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids. Plants and photosynthetic bacteria are uniquely able to convert CO2 to carbohydrates, using the Calvin cycle, as we shall see in Section 20.4. Although the reactions of gluconeogenesis are the same in all organisms, the metabolic context and the regulation of the pathway differ from one species to another and from tissue to tissue. In this section we focus on gluconeogenesis as it occurs in the mammalian liver. In Chapter 20 we show how photosynthetic organisms use this pathway to convert the primary products of photosynthesis into glucose, to be stored as sucrose or starch. Gluconeogenesis and glycolysis are not identical pathways running in opposite directions, although they do share several steps (Fig. 14-16); 7 of the 10 enzymatic reactions of gluconeogenesis are the reverse of glycolytic reactions. However, three reactions of glycolysis are essentially irreversible in vivo and cannot be used in gluconeogenesis: the conversion of glucose to glucose 6-phosphate by hexokinase, the phosphorylation of fructose 6-phosphate to fructose 1,6- bisphosphate by phosphofructokinase-1, and the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase. In cells, these three reactions are characterized by a large negative free-energy change, whereas other glycolytic reactions have a ΔG near 0 (Table 14-2). In gluconeogenesis, the three irreversible steps are bypassed by a separate set of enzymes, catalyzing reactions that are sufficiently exergonic to be effectively irreversible in the direction of glucose synthesis. Thus, both glycolysis and gluconeogenesis are irreversible processes in cells. In animals, both pathways occur largely in the cytosol, necessitating their reciprocal and coordinated regulation, described in Section 14.5. FIGURE 14-16 Opposing pathways of glycolysis and gluconeogenesis in liver. The reactions of glycolysis are on the le side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed in Section 14.5. TABLE 14-2 Free-Energy Changes of Glycolytic Reactions in Erythrocytes Glycolytic reaction step ΔG′° (kJ/mol) ΔG (kJ/mol) G lucose+ATP→ glucose6-phosphate+ADP −16.7 −33.4 G lucose6-phosphate ⇌  fructose6-phosphate 1.7 0 to 25 Fructose6-phosphate+ATP→  fructose1,6-bisphosphate+ADP −14.2 −22.2 Fructose1,6-bisphosphate ⇌  dihydroxyacetonephosphate+glyceraldehyde3-phosphate 23.8 −6to0 Dihydroxyacetone phosphate ⇌  glyceraldehyde 3-phosphate 7.5 0 to 4 G lyceraldehyde3-phosphate+Pi+NAD+ ⇌ 1,3-bisphosphoglycerate+NADH+H+ 6.3 −2to2 1,3-Bisphosphoglycerate+ADP ⇌ 3-phosphoglycerate+ATP −18.8 0 to 2 3-Phosphoglycerate ⇌ 2-phosphoglycerate 4.4 0 to 0.8 3-Phosphoglycerate ⇌  phosphoenolpyruvate+H2O 7.5 0 to 3.3 Phosphoenolpyruvate+ADP→ pyruvate+ATP −31.4 −16.7 Note: ΔG′° is the standard free-energy change, as defined in Chapter 13 (p. 468). ΔG is the free-energy change calculated from the actual concentrations of glycolytic intermediates present under physiological conditions in erythrocytes, at pH 7. The glycolytic reactions bypassed in gluconeogenesis are shown in red. Biochemical equations are not necessarily balanced for H or charge (p. 478). We begin by considering the three bypass reactions of gluconeogenesis. (Keep in mind that “bypass” refers throughout to the bypass of irreversible glycolytic reactions.) The First Bypass: Conversion of Pyruvate toPhosphoenolpyruvate Requires Two ExergonicReactions The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate (PEP). This reaction cannot occur by simple reversal of the pyruvate kinase reaction of glycolysis (p. 521), which has a large, negative free-energy change and is therefore irreversible under the conditions prevailing in intact cells (Table 14-2, step ). Instead, the phosphorylation of pyruvate is achieved by a roundabout sequence of reactions that in eukaryotes requires enzymes in both the cytosol and mitochondria. As we shall see, the pathway shown in Figure 14-16 and described in detail here is one of two routes from pyruvate to PEP; it is the predominant path when pyruvate or alanine is the glucogenic precursor. A second pathway, described later, predominates when lactate is the glucogenic precursor. Pyruvate is first transported from the cytosol into mitochondria or is generated from alanine within mitochondria by transamination, in which the α -amino group is transferred from alanine (leaving pyruvate) to an α -keto carboxylic acid (transamination reactions are discussed in detail in Chapter 18). Then pyruvate carboxylase, a mitochondrial enzyme that requires the coenzyme biotin, converts the pyruvate to oxaloacetate: Pyruvate+HCO−3+ATP→ oxaloacetate+ADP+Pi (14-4) The carboxylation reaction involves biotin as a carrier of activated bicarbonate, as shown in Figure 14-17; the reaction mechanism is shown in Figure 16-16. (Note that HCO−3 is formed by ionization of carbonic acid formed from CO2+H2O.) HCO−3 is phosphorylated by ATP to form a mixed anhydride (a carboxyphosphate); then biotin displaces the phosphate in the formation of carboxybiotin. FIGURE 14-17 Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to pyruvate carboxylase through an amide linkage to the ε -amino group of a Lys residue, forming a biotinyl-enzyme. The reaction takes place in two phases, which occur at two different sites in the enzyme. The long biotinyl-Lys arm carries the substrate from one site to the other. Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. Acetyl-CoA is produced by fatty acid oxidation (Chapter 17), and its accumulation signals the availability of fatty acids as fuel. As we shall see in Chapter 16, the pyruvate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle. Because the mitochondrial membrane has no transporter for oxaloacetate, before export to the cytosol the oxaloacetate formed from pyruvate must be reduced to malate by mitochondrial malate dehydrogenase, at the expense of NADH: Oxaloacetate+NADH+H+ ⇌ L-malate+NAD+ (14-5) The standard free-energy change for this reaction is quite high, but under physiological conditions (including a very low concentration of oxaloacetate) ΔG≈0 and the reaction is readily reversible. Mitochondrial malate dehydrogenase functions in both gluconeogenesis and the citric acid cycle, but the overall flow of metabolites in the two processes is in opposite directions. Malate leaves the mitochondrion through a specific transporter in the inner mitochondrial membrane (see Fig. 19-31), and in the cytosol it is reoxidized to oxaloacetate, with the production of cytosolic NADH: M alate+NAD+ → oxaloacetate+NADH+H+ (14-6) The oxaloacetate is then converted to PEP by phosphoenolpyruvate carboxykinase (Fig. 14-18). This M g2+-dependent reaction requires GTP as the phosphoryl group donor: Oxaloacetate+G TP ⇌  PEP+CO2+G DP (14-7) FIGURE 14-18 Synthesis of phosphoenolpyruvate from oxaloacetate. In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxykinase. The CO2 incorporated in the pyruvate carboxylase reaction is lost here as CO2. The decarboxylation leads to a rearrangement of electrons that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the γ phosphate of GTP. The reaction is reversible under intracellular conditions; the formation of one high- energy phosphate compound (PEP) is balanced by the hydrolysis of another (GTP). The overall equation for this set of bypass reactions, the sum of Equations 14-4 through 14-7, is Pyruvate+ATP+G TP+HCO−3 → PEP+ADP+G DP+Pi+CO2 ΔG′°=0.9 kJ/mol (14-8) Two high-energy phosphate equivalents (one from ATP and one from GTP), each yielding about 50 kJ/mol under cellular conditions, must be expended to phosphorylate one molecule of pyruvate to PEP. In contrast, when PEP is converted to pyruvate during glycolysis, only one ATP is generated from ADP. Although the standard free-energy change (ΔG′°) of the two-step path from pyruvate to PEP is 0.9 kJ/mol, the actual free-energy change (ΔG), calculated from measured cellular concentrations of intermediates, is very strongly negative (−25kJ/mol); this results from the ready consumption of PEP in other reactions such that its concentration remains relatively low. The reaction is thus effectively irreversible in the cell. Note that the CO2 added to pyruvate in the pyruvate carboxylase step (Fig. 14-17) is the same molecule that is lost in the PEP carboxykinase reaction (Fig. 14-18). This carboxylation-decarboxylation sequence represents a way of “activating” pyruvate, in that the decarboxylation of oxaloacetate facilitates PEP formation. In Chapter 21 we shall see how a similar carboxylation-decarboxylation sequence is used to activate acetyl-CoA for fatty acid biosynthesis (see Fig. 21-1). There is a logic to the route of these reactions through the mitochondrion. The [NADH]/[NAD+] ratio in the cytosol is several orders of magnitude lower than in mitochondria. Because cytosolic NADH is consumed in gluconeogenesis (in the conversion of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate; Fig. 14-16), glucose biosynthesis cannot proceed unless NADH is available. The transport of malate from the mitochondrion to the cytosol and its reconversion there to oxaloacetate effectively moves reducing equivalents to the cytosol, where they are scarce. This path from pyruvate to PEP therefore provides an important balance between NADH produced and consumed in the cytosol during gluconeogenesis. A second pyruvate → PEP bypass predominates when lactate is the glucogenic precursor (Fig. 14-19). This pathway makes use of lactate produced by glycolysis in erythrocytes or anaerobic muscle, for example, and it is particularly important in large vertebrates aer vigorous exercise (Box 14-2). The conversion of lactate to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria is therefore unnecessary. Aer the pyruvate produced by the lactate dehydrogenase reaction is transported into the mitochondrion (by a transporter in the inner mitochondrial membrane specific for pyruvate), it is converted to oxaloacetate by pyruvate carboxylase, as described above. This oxaloacetate, however, is converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase, and the PEP is transported out of the mitochondrion to continue on the gluconeogenic path. The mitochondrial and cytosolic isozymes of PEP carboxykinase are encoded by separate genes in the nuclear chromosomes, providing another example of two distinct enzymes catalyzing the same reaction but having different cellular locations or metabolic roles (recall the isozymes of hexokinase).

FIGURE 14-19 Alternative paths from pyruvate to phosphoenolpyruvate. The relative importance of the two pathways depends on the availability of lactate or pyruvate and the cytosolic requirements for NADH for gluconeogenesis. The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion (see text). The Second and Third Bypasses Are SimpleDephosphorylations by Phosphatases The second glycolytic reaction that cannot participate in gluconeogenesis is the phosphorylation of fructose 6-phosphate by PFK-1 (Table 14-2, step ). Because this reaction is highly exergonic and therefore irreversible in intact cells, the generation of fructose 6-phosphate from fructose 1,6-bisphosphate (Fig. 14-16) is catalyzed by a different enzyme, M g2+-dependent fructose 1,6-bisphosphatase (FBPase-1), which promotes the essentially irreversible hydrolysis of the C-1 phosphate (not phosphoryl group transfer to ADP): Fructose1,6-phosphate+H2O→ fructose6-phosphate+Pi ΔG′°=−16.3kJ/mol FBPase-1 is so named to distinguish it from another, similar enzyme (FBPase-2) with a regulatory role, which we discuss in Section 14.5. The third bypass is the final reaction of gluconeogenesis, the dephosphorylation of glucose 6-phosphate to yield glucose (Fig. 14-16). Reversal of the hexokinase reaction (p. 514) would require phosphoryl group transfer from glucose 6-phosphate to ADP, forming ATP, an energetically unfavorable reaction (Table 14-2, step ). The reaction catalyzed by glucose 6-phosphatase does not require synthesis of ATP; it is a simple hydrolysis of a phosphate ester: G lucose6-phosphate+H2O→glucose+Pi ΔG′°=−13.8kJ/mol This M g2+-activated enzyme is a membrane protein in the lumen of the endoplasmic reticulum of hepatocytes, renal cells, and epithelial cells of the small intestine (see Fig. 15-6), but not in other tissues, which are therefore unable to supply glucose to the blood. If other tissues had glucose 6-phosphatase, this enzyme’s activity would hydrolyze the glucose 6-phosphate needed within those tissues for glycolysis. Glucose produced by gluconeogenesis in the liver or kidney or ingested in the diet is delivered to these other tissues, including brain and muscle, through the bloodstream. Gluconeogenesis Is Energetically Expensive, ButEssential The sum of the biosynthetic reactions leading from pyruvate to free blood glucose (Table 14-3) is 2Pyruvate+4ATP+2G TP+2NADH+2H+ +4H2O→ glucose+4ADP+2G DP+6Pi+2NAD+ (14-9) TABLE 14-3 Sequential Reactions in Gluconeogenesis Starting from Pyruvate Pyruvate+HCO−3+ATP→ oxaloacetate+ADP+Pi ×2 Oxaloacetate+G TP⇌ phosphoenolpyruvate+CO2+G DP ×2 Phosphoenolpyruvate+H2O⇌ 2-phosphoglycerate ×2 2-Phosphoglycerate⇌ 3-phosphoglycerate ×2 3-Phosphoglycerate+ATP⇌ 1,3-bisphosphoglycerate+ADP ×2 1,3-Bisphosphoglycerate+NADH+H+ ⇌ glyceraldehyde3-phosphate+NAD+ +Pi ×2 G lyceraldehyde3-phosphate⇌ dihydroxyacetonephosphate G lyceraldehyde3-phosphate+dihydroxyacetonephosphate⇌ fructose1,6-bisphosphate Fructose1,6-bisphosphate→ fructose6-phosphate+Pi Fructose6-phosphate⇌ glucose6-phosphate G lucose6-phosphate+H2O→ glucose+Pi Sum:2Pyruvate+4ATP+2G TP+2NADH+2H+ +4H2O→ glucose+4ADP+2G DP+6Pi+2NAD+ Note: The bypass reactions are in red; all other reactions are reversible steps of glycolysis. The figures at the right indicate that the reaction is to be counted twice, because two three-carbon precursors are required to make a molecule of glucose. The reactions required to replace the cytosolic NADH consumed in the glyceraldehyde 3-phosphate dehydrogenase reaction (the conversion of lactate to pyruvate in the cytosol or the transport of reducing equivalents from mitochondria to the cytosol in the form of malate) are not considered in this summary. Biochemical equations are not necessarily balanced for H and charge (p. 478). For each molecule of glucose formed from pyruvate, six high-energy phosphate groups are required: four from ATP and two from GTP. In addition, two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate. Clearly, Equation 14-9 is not simply the reverse of the equation for conversion of glucose to pyruvate by glycolysis, which would require only two molecules of ATP: G lucose+2ADP+2Pi+NAD+ → 2pyruvate+2ATP+2NADH+2H+ +2H2O This makes the synthesis of glucose from pyruvate a relatively expensive process. Much of this high energy cost is necessary to ensure the irreversibility of gluconeogenesis. Under intracellular conditions, the overall free-energy change of glycolysis is at least −63kJ/mol. Under the same conditions the overall ΔG of gluconeogenesis is −16kJ/mol. Thus both glycolysis and gluconeogenesis are essentially irreversible processes in cells. A second advantage to investing energy to convert pyruvate to glucose is that if pyruvate were instead excreted, its considerable potential for ATP production by complete, aerobic oxidation would be lost (more than 10 ATP are produced per pyruvate, as we shall see in Chapter 16). The biosynthetic pathway to glucose described above allows the net synthesis of glucose not only from pyruvate but also from the four-, five-, and six-carbon intermediates of the citric acid cycle (Chapter 16). The citric acid cycle intermediates can undergo oxidation to oxaloacetate (see Fig. 16-7). Some or all of the carbon atoms of most amino acids derived from proteins are ultimately catabolized to pyruvate or to intermediates of the citric acid cycle. Such amino acids can therefore undergo net conversion to glucose and are said to be glucogenic (Table 14-4). Alanine and glutamine, the principal molecules that transport amino groups from extrahepatic tissues to the liver (see Fig. 18-9), are particularly important glucogenic amino acids in mammals. Aer removal of their amino groups in liver mitochondria, the carbon skeletons remaining (pyruvate and α -ketoglutarate, respectively) are readily funneled into gluconeogenesis. TABLE 14-4 Glucogenic Amino Acids, Grouped by Site of Entry Pyruvate Succinyl-CoA Alanine Isoleucine Cysteine Methionine Glycine Threonine Serine Valine Threonine Fumarate Tryptophan Phenylalanine α -Ketoglutarate Tyrosine Arginine Oxaloacetate Glutamate Asparagine Glutamine Aspartate Histidine Proline Note: All these amino acids are precursors of blood glucose or liver glycogen, because they can be converted to pyruvate or citric acid cycle intermediates. Of the 20 common amino acids, only leucine and lysine are unable to furnish carbon for net glucose synthesis. These amino acids are also ketogenic (see Fig. 18-15). Mammals Cannot Convert Fatty Acids toGlucose; Plants and Microorganisms Can No net conversion of fatty acids to glucose occurs in mammals. As we shall see in Chapter 17, the catabolism of most fatty acids yields only acetyl-CoA. Mammals a a a a a cannot use acetyl-CoA as a precursor of glucose, because the pyruvate dehydrogenase reaction is irreversible and cells have no other pathway to convert acetyl-CoA to pyruvate. Plants, yeast, and many bacteria do have a pathway (the glyoxylate cycle; see Fig. 20-45) for converting acetyl-CoA to oxaloacetate, so these organisms can use fatty acids as the starting material for gluconeogenesis. This is important during the germination of seedlings, for example; before leaves develop and photosynthesis can provide energy and carbohydrates, the seedling relies on stored seed oils for energy production and cell wall biosynthesis. Although mammals cannot convert fatty acids to carbohydrate, they can use the small amount of glycerol produced from the breakdown of fats (triacylglycerols) for gluconeogenesis. Phosphorylation of glycerol by glycerol kinase, followed by oxidation of the central carbon, yields dihydroxyacetone phosphate, an intermediate in gluconeogenesis in liver. As we shall see in Chapter 21, glycerol phosphate is an essential intermediate in triacylglycerol synthesis in adipocytes, but these cells lack glycerol kinase and so cannot simply phosphorylate glycerol. Instead, adipocytes carry out a truncated version of gluconeogenesis, known as glyceroneogenesis: the conversion of pyruvate to dihydroxyacetone phosphate via the early reactions of gluconeogenesis, followed by reduction of the dihydroxyacetone phosphate to glycerol 3-phosphate (see Fig. 21-21). SUMMARY 14.4 Gluconeogenesis Gluconeogenesis is a multistep process in which glucose is produced from lactate, pyruvate, or oxaloacetate, or any compound (including citric acid cycle intermediates) that can be converted to one of these intermediates. Seven steps are the reversal of glycolytic reactions; three differ and must be bypassed with exergonic reactions. In the first bypass, pyruvate is converted to PEP via oxaloacetate in two steps catalyzed by pyruvate carboxylase (which uses ATP) and PEP carboxykinase (which uses GTP). In the second bypass, FBPase-1 removes a phosphate group from fructose 1,6- bisphosphate, producing fructose 6-phosphate. In the third bypass, glucose 6- phosphatase converts glucose 6-phosphate to glucose. In mammals, gluconeogenesis in the liver, kidney, and small intestine provides glucose for use by the brain, muscles, and erythrocytes. Formation of one molecule of glucose from pyruvate requires four ATP, two GTP, and two NADH; it is expensive. Animals cannot convert acetyl-CoA derived from fatty acids into glucose; they lack the enzymatic machinery to convert acetyl-CoA to pyruvate. Plants and microorganisms have the glyoxylate pathway, which allows them to make glucose from fatty acids. 14.5 Coordinated Regulation of Glycolysis and Gluconeogenesis Glycolysis (the conversion of glucose to pyruvate) and gluconeogenesis (the conversion of pyruvate to glucose) generally do not both occur at the same time in the same tissues. In mammals, gluconeogenesis occurs primarily in the liver, where its role is to provide glucose for export to other tissues when glycogen stores are exhausted and when no dietary glucose is available. Glycolysis occurs in most tissues, including, brain, kidney, muscle and liver. Glycolysis provides ATP to support all of the energy-requiring activities of cells: active transport of ions; synthesis of macromolecules and of their precursors; synthesis of lipids and storage compounds like glycogen; and muscle contraction. At each of the three points where glycolytic reactions are bypassed by alternative, gluconeogenic reactions (Fig. 14-16), simultaneous operation of both pathways would consume ATP without accomplishing any chemical or biological work. For example, PFK-1 and FBPase-1 catalyze opposing reactions: The sum of these two reactions is ATP + H2O → ADP + Pi+ heat that is, hydrolysis of ATP without any useful metabolic work being done. Clearly, if these two reactions were allowed to proceed simultaneously at a high rate in the same cell, a large amount of chemical energy would be dissipated as heat. We look now in some detail at the mechanisms that regulate glycolysis and gluconeogenesis at the three points where these pathways diverge. Hexokinase Isozymes Are Affected Differently by Their Product, Glucose 6-Phosphate Hexokinase, which catalyzes the entry of glucose into the glycolytic pathway, is a regulatory enzyme. As noted in Section 14.1, humans have four isozymes of hexokinase (designated I to IV), encoded by four different genes (Box 14-3). The different hexokinase isozymes of liver and muscle reflect the different roles of these organs in carbohydrate metabolism: muscle consumes glucose, using it for energy production; liver maintains blood glucose homeostasis by consuming or producing glucose, depending on the prevailing blood glucose concentration. BOX 14-3 Isozymes: Different Proteins That Catalyze the Same Reaction The four forms of hexokinase found in mammalian tissues are but one example of a common biological situation: the same reaction catalyzed by two or more different molecular forms of an enzyme. These multiple forms, called isozymes or isoenzymes, may occur in the same species, in the same tissue, even in the same cell. The different forms (isoforms) of the enzyme generally differ in kinetic or regulatory properties, in the cofactor they use (NADH or NADPH for dehydrogenase isozymes, for example), or in their subcellular distribution (soluble or membrane-bound). Isozymes may have similar, but not identical, amino acid sequences, and in many cases they clearly share a common evolutionary origin. One of the first enzymes found to have isozymes was lactate dehydrogenase (LDH; p. 526), which in vertebrate tissues exists as at least five different isozymes separable by electrophoresis. All LDH isozymes contain four polypeptide chains (each of Mr 33,500), each type containing a different ratio of two kinds of polypeptides. The M (for muscle) chain and the H (for heart) chain are encoded by two different genes. In skeletal muscle the predominant isozyme contains four M chains, and in heart the predominant isozyme contains four H chains. Other tissues have some combination of the five possible types of LDH isozymes: Type Composition Location LDH1 HHHH Heart and erythrocyte LDH2 HHHM Heart and erythrocyte LDH3 HHMM Brain and kidney LDH4 HMMM Skeletal muscle and liver LDH5 MMMM Skeletal muscle and liver Differences in the isozyme content of tissues can be used to assess the timing and extent of heart damage due to myocardial infarction (heart attack). Damage to heart tissue results in the release of heart LDH into the blood. Shortly a er a heart attack, the blood level of total LDH increases, and there is more LDH2 than LDH1. A er 12 hours the amounts of LDH1 and LDH2 are very similar, and a er 24 hours there is more LDH1 than LDH2. This switch in the [LDH1]/[LDH2] ratio, combined with increased concentrations in the blood of another heart enzyme, creatine kinase, is very strong evidence of a recent myocardial infarction. The different LDH isozymes have significantly different values of Vmax and Km, particularly for pyruvate. The properties of LDH4 favor rapid reduction of very low concentrations of pyruvate to lactate in skeletal muscle, whereas those of isozyme LDH1 favor rapid oxidation of lactate to pyruvate in the heart. In general, the distribution of different isozymes of a given enzyme reflects at least four factors: 1. Different metabolic patterns in different organs. For glycogen phosphorylase, the isozymes in skeletal muscle and liver have different regulatory properties, reflecting the different roles of glycogen breakdown in these two tissues. 2. Different locations and metabolic roles for isozymes in the same cell. The isocitrate dehydrogenase isozymes of the cytosol and the mitochondrion are an example (Chapter 16). 3. Different stages of development in embryonic or fetal tissues and in adult tissues. For example, the fetal liver has a characteristic isozyme distribution of LDH, which changes as the organ develops into its adult form. Some enzymes of glucose catabolism in malignant (cancer) cells occur as their fetal, not adult, isozymes. 4. Different responses of isozymes to allosteric modulators. This difference is useful in fine-tuning metabolic rates. Hexokinase IV (glucokinase) of liver and the hexokinase isozymes of other tissues differ in their sensitivity to inhibition by glucose 6-phosphate. The predominant hexokinase isozyme of myocytes (hexokinase II) has a high affinity for glucose — it is half-saturated at about 0.1 mM. Because glucose entering myocytes from the blood (where the glucose concentration is 4 to 5 mM) produces an intracellular glucose concentration high enough to saturate hexokinase II, the muscle enzyme normally acts at or near its Vmax. Muscle hexokinase I and hexokinase II are allosterically inhibited by their product, glucose 6-phosphate, so whenever the cellular concentration of glucose 6-phosphate rises above its normal level, these isozymes are temporarily and reversibly inhibited, bringing the rate of glucose 6-phosphate formation into balance with the rate of its utilization and reestablishing the steady state. The predominant hexokinase isozyme of liver is hexokinase IV (also called glucokinase), which differs in three important respects from hexokinases I, II, and III of muscle. First, the glucose concentration at which hexokinase IV is half-saturated (about 10 mM) is higher than the usual concentration of glucose in the blood. Because an efficient glucose transporter in hepatocytes (GLUT2) rapidly equilibrates the glucose concentrations in cytosol and blood, the high Km of hexokinase IV allows its direct regulation by the blood glucose concentration (Fig. 14-20). When blood glucose is high, as it is aer a meal rich in carbohydrates, excess glucose is transported into hepatocytes, where hexokinase IV converts it to glucose 6-phosphate. Because hexokinase IV is not saturated at 10 mM glucose, its activity continues to increase as the glucose concentration rises to 10 mM or more. Under conditions of low blood glucose, the glucose concentration in a hepatocyte is low relative to the Km of hexokinase IV, and the glucose generated by gluconeogenesis leaves the cell before being trapped by phosphorylation. FIGURE 14-20 Comparison of the kinetic properties of hexokinase IV (glucokinase) and hexokinase I. Note the much lower Km for hexokinase I. When blood glucose rises above 5 m , hexokinase IV activity increases, but hexokinase I is already operating near Vmax and cannot respond to an increase in glucose concentration. Hexokinases I, II, and III have similar kinetic properties. Second, hexokinase IV is not inhibited by glucose 6-phosphate, and it can therefore continue to operate when the accumulation of glucose 6-phosphate completely inhibits hexokinases I, II, and III. Third, hexokinase IV is subject to inhibition by the reversible binding of a regulatory protein specific to liver (Fig. 14-21). The binding is much tighter in the presence of the allosteric effector fructose 6-phosphate. Glucose competes with fructose 6- phosphate for binding and causes dissociation of the regulatory protein from the hexokinase, relieving the inhibition. Immediately aer a carbohydrate-rich meal, when blood glucose is high, glucose enters the hepatocyte via GLUT2 and activates hexokinase IV by this mechanism. During a fast, when blood glucose drops below 5 mM, fructose 6-phosphate triggers the inhibition of hexokinase IV by the regulatory protein, so the liver does not compete with other organs for the scarce glucose. The mechanism of inhibition by the regulatory protein is interesting: the protein anchors hexokinase IV inside the nucleus, where it is segregated from the other enzymes of glycolysis in the cytosol. When the glucose concentration in the cytosol rises, it equilibrates with glucose in the nucleus by transport through the nuclear pores. Glucose causes dissociation of the regulatory protein, and hexokinase IV enters the cytosol and begins to phosphorylate glucose. FIGURE 14-21 Regulation of hexokinase IV (glucokinase) by sequestration in the nucleus. The protein inhibitor of hexokinase IV is a nuclear binding protein that draws hexokinase IV into the nucleus when the fructose 6- phosphate concentration in liver is high and releases it to the cytosol when the glucose concentration is high. Hexokinase IV is also regulated at the level of protein synthesis. Circumstances that call for greater energy production (low [ATP], high [AMP], vigorous muscle contraction) or for greater glucose consumption (high blood [glucose], for example) cause increased transcription of the hexokinase IV gene. Glucose 6-phosphatase, the gluconeogenic enzyme that bypasses the hexokinase step of glycolysis, is transcriptionally regulated by factors that call for increased production of glucose (low blood glucose, glucagon signaling). The transcriptional regulation of these two enzymes (along with other enzymes of glycolysis and gluconeogenesis) is described below. Phosphofructokinase-1 and Fructose 1,6-Bisphosphatase Are Reciprocally Regulated Glucose 6-phosphate can flow either into glycolysis or through any of several other pathways, including glycogen synthesis and the pentose phosphate pathway. The metabolically irreversible reaction catalyzed by PFK-1 is the step that commits glucose to glycolysis. In addition to its substrate-binding sites, this complex enzyme has several regulatory sites at which allosteric activators or inhibitors bind. ATP is not only a substrate for PFK-1 but also an end product of the glycolytic pathway. When high cellular [ATP] signals that ATP is being produced faster than it is being consumed, ATP inhibits PFK-1 by binding to an allosteric site and lowering the affinity of the enzyme for its substrate fructose 6-phosphate (Fig. 14-22). ADP and AMP, which increase in concentration as consumption of ATP outpaces production, act allosterically to relieve this inhibition by ATP. These effects combine to produce higher enzyme activity when ADP or AMP accumulates and lower activity when ATP accumulates.

FIGURE 14-22 Phosphofructokinase-1 (PFK-1) and its regulation. (a) Surface contour image of E. coli PFK-1, showing portions of its four identical subunits. Each subunit has its own catalytic site, where the products ADP and fructose 1,6-bisphosphate (red and yellow stick structures, respectively) are almost in contact, and its own binding sites for the allosteric regulator ATP, buried in the protein in the positions indicated. (b) Allosteric regulation of muscle PFK-1 by ATP, shown by a substrate-activity curve. At low [ATP], the K0.5 for fructose 6-phosphate is relatively low, enabling the enzyme to function at a high rate at relatively low [fructose 6- phosphate]. (Recall from Chapter 6 that K0.5 is the Km term for regulatory enzymes; when K0.5 is larger, the binding is weaker.) When [ATP] is high, K0.5 for fructose 6-phosphate is greatly increased, as indicated by the sigmoid relationship between substrate concentration and enzyme activity. (c) Summary of the regulators affecting PFK-1 activity. [(a) Data from PDB ID 1PFK, Y. Shirakihara and P. R. Evans, J. Mol. Biol. 204:973, 1988.] Citrate (the ionized form of citric acid), a key intermediate in the aerobic oxidation of pyruvate, fatty acids, and amino acids, is also an allosteric regulator of PFK-1. High citrate concentration increases the inhibitory effect of ATP, further reducing the flow of glucose through glycolysis. In this case, as in several others encountered later, citrate serves as an intracellular signal that the cell is meeting its current needs for energy-yielding metabolism by the oxidation of fats and proteins. The corresponding step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate (Fig. 14-23). The enzyme that catalyzes this reaction, FBPase-1, is strongly inhibited (allosterically) by AMP; when the cell’s supply of ATP is low (corresponding to high [AMP]), the ATP-requiring synthesis of glucose slows. FIGURE 14-23 Regulation of phosphofructokinase-1 (PFK-1) and fructose 1,6-bisphosphatase (FBPase-1). Thus, these opposing steps in the glycolytic and gluconeogenic pathways — those catalyzed by PFK-1 and FBPase-1 — are regulated in a coordinated and reciprocal manner. In general, when sufficient concentrations of acetyl-CoA or citrate (the product of acetyl-CoA condensation with oxaloacetate) are present, or when a high proportion of the cell’s adenylate is in the form of ATP, gluconeogenesis is favored. When the concentration of AMP increases, it promotes glycolysis by stimulating PFK-1 (and, as we shall see in Section 15.3, promotes glycogen degradation by activating glycogen phosphorylase). Fructose 2,6-Bisphosphate Is a Potent Allosteric Regulator of PFK-1 and FBPase-1 The special role of the liver in maintaining a constant blood glucose level requires additional regulatory mechanisms to coordinate glucose production and consumption. When the blood glucose level decreases, the hormone glucagon signals the liver to produce and release more glucose and to stop consuming it for its own needs. One source of glucose is glycogen stored in the liver; another source is gluconeogenesis, using pyruvate, lactate, glycerol, or certain amino acids as starting material. When blood glucose is high, insulin signals the liver to use glucose as a fuel and as a precursor for the synthesis and storage of glycogen and triacylglycerol. The rapid hormonal regulation of glycolysis and gluconeogenesis is mediated by fructose 2,6-bisphosphate, an allosteric effector for the enzymes PFK-1 and FBPase-1: When fructose 2,6-bisphosphate binds to its allosteric site on PFK-1, it increases the enzyme’s affinity for its substrate fructose 6-phosphate (Fig. 14-24a) and reduces its affinity for the allosteric inhibitors ATP and citrate. At the physiological concentrations of its substrates, ATP and fructose 6-phosphate, and of its other positive and negative effectors (ATP, AMP, citrate), PFK-1 is virtually inactive in the absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate has the opposite effect on FBPase-1: it reduces its affinity for its substrate (Fig. 14-24b), thereby slowing gluconeogenesis. FIGURE 14-24 Role of fructose 2,6-bisphosphate in regulation of glycolysis and gluconeogenesis. Fructose 2,6-bisphosphate (F26BP) has opposite effects on the enzymatic activities of phosphofructokinase-1 (PFK-1, a glycolytic enzyme) and fructose 1,6-bisphosphatase (FBPase-1, a gluconeogenic enzyme). (a) PFK-1 activity in the absence of F26BP (blue curve) is half-maximal when the concentration of fructose 6- phosphate is 2 m (that is, K0.5= 2mM). When 0.13 μM F26BP is present (red curve), the K0.5 for fructose 6-phosphate is only 0.08 m . Thus F26BP activates PFK-1 by increasing its apparent affinity for fructose 6-phosphate (see Fig. 14-23b). (b) FBPase-1 activity is inhibited by as little as 1μM F26BP and is strongly inhibited by 25 μM. In the absence of this inhibitor (blue curve), the K0.5 for fructose 1,6-bisphosphate is 5μM, but in the presence of 25μM F26BP (red curve), the K0.5 is > 70μM. Fructose 2,6- bisphosphate also makes FBPase-1 more sensitive to inhibition by another allosteric regulator, AMP. (c) Summary of regulation by F26BP. The cellular concentration of the allosteric regulator fructose 2,6- bisphosphate is set by the relative rates of its formation and breakdown (Fig. 14-25a). It is formed by phosphorylation of fructose 6-phosphate, catalyzed by phosphofructokinase-2 (PFK- 2), and broken down by fructose 2,6-bisphosphatase (FBPase-2). (Note that these enzymes are distinct from PFK-1 and FBPase-1, which catalyze the formation and breakdown, respectively, of fructose 1,6-bisphosphate.) PFK-2 and FBPase-2 are two separate enzymatic activities of a single, bifunctional protein. The balance of these two activities in the liver, which determines the cellular level of fructose 2,6-bisphosphate, is set by glucagon and insulin (Fig. 14-25b). FIGURE 14-25 Regulation of fructose 2,6-bisphosphate level. (a) The cellular concentration of the regulator fructose 2,6-bisphosphate (F26BP) is determined by the rates of its synthesis by phosphofructokinase-2 (PFK-2) and its breakdown by fructose 2,6-bisphosphatase (FBPase-2). (b) Both enzyme activities are part of the same polypeptide chain, and they are reciprocally regulated by insulin and glucagon. As we saw in Chapter 12, glucagon stimulates the adenylyl cyclase of liver to synthesize 3′,5′-cyclic AMP (cAMP) from ATP. Cyclic AMP then activates cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the bifunctional protein PFK-2/FBPase-2. Phosphorylation of this protein enhances its FBPase-2 activity and inhibits its PFK-2 activity. Glucagon thereby lowers the cellular level of fructose 2,6- bisphosphate, inhibiting glycolysis and stimulating gluconeogenesis. The resulting production of more glucose enables the liver to replenish blood glucose in response to glucagon. Insulin has the opposite effect, stimulating the activity of a phosphoprotein phosphatase that catalyzes removal of the phosphoryl group from the bifunctional protein PFK-2/FBPase-2, activating its PFK-2 activity, increasing the level of fructose 2,6- bisphosphate, stimulating glycolysis, and inhibiting gluconeogenesis. Xylulose 5-Phosphate Is a Key Regulator of Carbohydrate and Fat Metabolism Another regulatory mechanism also acts by controlling the level of fructose 2,6-bisphosphate. In the mammalian liver, xylulose 5- phosphate, a product of the pentose phosphate pathway, mediates the increase in glycolysis that follows ingestion of a high- carbohydrate meal. The xylulose 5-phosphate concentration rises as glucose entering the liver is converted to glucose 6-phosphate and enters both the glycolytic and pentose phosphate pathways. Xylulose 5-phosphate activates phosphoprotein phosphatase 2A, which dephosphorylates the bifunctional PFK-2/FBPase-2 enzyme (Fig. 14-25). Dephosphorylation activates PFK-2 and inhibits FBPase-2, and the resulting rise in fructose 2,6-bisphosphate concentration stimulates glycolysis and inhibits gluconeogenesis. The increased glycolysis boosts the production of acetyl-CoA, while the increased flow of hexose through the pentose phosphate pathway generates NADPH. Acetyl-CoA and NADPH are the starting materials for fatty acid synthesis, which increases dramatically in response to intake of a high-carbohydrate meal. Xylulose 5-phosphate also increases the synthesis of all the enzymes required for fatty acid synthesis, as we shall see (Fig. 14- 28). The Glycolytic Enzyme Pyruvate Kinase Is Allosterically Inhibited by ATP At least three isozymes of pyruvate kinase are found in vertebrates, differing in their tissue distribution and their response to modulators. High concentrations of ATP, acetyl-CoA, and long-chain fatty acids (signs of abundant energy supply) allosterically inhibit all isozymes of pyruvate kinase (Fig. 14-26). The liver isozyme (L form), but not the muscle isozyme (M form), is subject to further regulation by phosphorylation. When low blood glucose causes glucagon release, cAMP-dependent protein kinase phosphorylates the L isozyme of pyruvate kinase, inactivating it. This slows the use of glucose as a fuel in liver, sparing it for export to the brain and other organs. In muscle, the effect of increased [cAMP] is quite different. In response to epinephrine, cAMP activates glycogen breakdown and glycolysis and provides the fuel needed for the fight-or-flight response. FIGURE 14-26 Regulation of pyruvate kinase. The enzyme is allosterically inhibited by ATP, acetyl-CoA, and long-chain fatty acids (all signs of an abundant energy supply), and the accumulation of fructose 1,6-bisphosphate triggers its activation. Accumulation of alanine, which can be synthesized from pyruvate in one step, allosterically inhibits pyruvate kinase, slowing the production of pyruvate by glycolysis. The liver isozyme (L form) is also regulated hormonally. Glucagon activates cAMP-dependent protein kinase (PKA; see Fig. 15-12), which phosphorylates the pyruvate kinase L isozyme, inactivating it. When the glucagon level drops, a protein phosphatase (PP) dephosphorylates pyruvate kinase, activating it. This mechanism prevents the liver from consuming glucose by glycolysis when blood glucose is low; instead, the liver exports glucose. The muscle isozyme (M form) is not affected by this phosphorylation mechanism. Conversion of Pyruvate to Phosphoenolpyruvate Is Stimulated When Fatty Acids Are Available In the pathway leading from pyruvate to glucose, the first control point determines the fate of pyruvate in the mitochondrion: its conversion either to acetyl-CoA (by the pyruvate dehydrogenase complex) to fuel the citric acid cycle (Chapter 16) or to oxaloacetate (by pyruvate carboxylase) to start the process of gluconeogenesis (Fig. 14-27). When fatty acids are readily available as fuels, their breakdown in liver mitochondria yields acetyl-CoA, a signal that further oxidation of glucose for fuel is not necessary. Acetyl-CoA is a positive allosteric modulator of pyruvate carboxylase and a negative modulator of pyruvate dehydrogenase, through stimulation of a protein kinase that inactivates the dehydrogenase. When the cell’s energy needs are being met, oxidative phosphorylation slows, [NADH] rises relative to [NAD+] and inhibits the citric acid cycle, and acetyl-CoA accumulates. The increased concentration of acetyl-CoA inhibits the pyruvate dehydrogenase complex, slowing the formation of acetyl-CoA from pyruvate, and stimulates gluconeogenesis by activating pyruvate carboxylase, allowing conversion of excess pyruvate to oxaloacetate (and, eventually, glucose).

FIGURE 14-27 Two alternative fates for pyruvate. Pyruvate can be converted to glucose and glycogen via gluconeogenesis or oxidized to acetyl-CoA for energy production. The first enzyme in each path is regulated allosterically; acetyl-CoA, produced either by fatty acid oxidation or by the pyruvate dehydrogenase complex, stimulates pyruvate carboxylase and inhibits pyruvate dehydrogenase. Oxaloacetate formed in this way is converted to phosphoenolpyruvate (PEP) in the reaction catalyzed by PEP carboxykinase (Fig. 14-16). In mammals, the regulation of this key enzyme occurs primarily at the level of its synthesis and breakdown, in response to dietary and hormonal signals. Fasting or high glucagon levels act through cAMP to increase the rate of transcription and to stabilize the mRNA. Insulin, or high blood [glucose], has the opposite effects. We discuss this transcriptional regulation in more detail below. Generally triggered by a signal from outside the cell, these changes take place on a time scale of minutes to days. Transcriptional Regulation Changes the Number of Enzyme Molecules Most of the regulatory actions discussed thus far are mediated by fast, reversible mechanisms to change the activity of existing enzyme molecules: allosteric effects, covalent alteration (phosphorylation) of the enzyme, or binding of a regulatory protein. Another set of regulatory processes involves changes in the number of molecules of an enzyme in the cell, through changes in the balance of enzyme synthesis and breakdown. Our discussion now turns briefly to regulation of transcription through signal-activated transcription factors. Transcriptional control is discussed in more detail in Chapter 28. In Chapter 12 we encountered nuclear receptors and transcription factors in the context of insulin signaling. Insulin acts through its receptor in the plasma membrane to turn on at least two distinct signaling pathways, each involving activation of a protein kinase (MAP kinase and protein kinase B). The kinases phosphorylate transcription factors, which then act in the nucleus to stimulate the synthesis of enzymes needed for cell growth and division. More than 150 genes are transcriptionally regulated by insulin, many of which encode proteins we have described here (Table 14-5). TABLE 14-5 Some of the Many Genes Regulated by Insulin Change in gene expression Role in glucose metabolism Increased expression Hexokinase II Hexokinase IV Phosphofructokinase-1 (PFK-1) PFK-2/FBPase-2 Pyruvate kinase Essential for glycolysis, which consumes glucose for energy Glucose 6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Malic enzyme Produce NADPH, which is essential for conversion of glucose to lipids ATP-citrate lyase Pyruvate dehydrogenase Produce acetyl-CoA, which is essential for conversion of glucose to lipids Acetyl-CoA carboxylase Fatty acid synthase complex Stearoyl-CoA dehydrogenase Acyl-CoA–glycerol transferases Essential for conversion of glucose to lipids Decreased expression PEP carboxykinase Glucose 6-phosphatase (catalytic subunit) Essential for glucose production by gluconeogenesis One transcription factor important to carbohydrate metabolism is ChREBP (carbohydrate response element binding protein; Fig. 14-28), which is expressed primarily in liver, adipose tissue, and kidney. It coordinates the synthesis of enzymes needed for carbohydrate and fat synthesis. ChREBP in its phosphorylated form is inactive and is located in the cytosol. When the phosphoprotein phosphatase PP2A removes a phosphoryl group from ChREBP, the transcription factor can enter the nucleus. Here, nuclear PP2A removes another phosphoryl group, and ChREBP now joins with a partner protein, Mlx, and turns on the synthesis of several enzymes: pyruvate kinase; fatty acid synthase; and acetyl-CoA carboxylase, the first enzyme in the path to fatty acid synthesis. FIGURE 14-28 Mechanism of gene regulation by the transcription factor ChREBP. When ChREBP in the cytosol of a hepatocyte is phosphorylated on a Ser residue and a Thr residue, it cannot enter the nucleus. Dephosphorylation of —Ser by protein phosphatase PP2A allows ChREBP to enter the nucleus, where a second dephosphorylation, of — Thr, activates ChREBP so that it can associate with its partner protein, Mlx. ChREBP-Mlx now binds to the carbohydrate response element (ChoRE) in the promoter and stimulates transcription. PP2A is allosterically activated by xylulose 5-phosphate, an intermediate in the pentose phosphate pathway. Controlling the activity of PP2A — and thus, ultimately, the synthesis of this group of metabolic enzymes — is xylulose 5- phosphate, an intermediate of the pentose phosphate pathway (see Fig. 14-31). When blood glucose concentration is high, glucose enters the liver and is phosphorylated by hexokinase IV. The glucose 6-phosphate thus formed can enter either the glycolytic pathway or the pentose phosphate pathway. If the latter, two initial oxidations produce xylulose 5-phosphate, which serves as a signal that the glucose-utilizing pathways are well-supplied with substrate. It accomplishes this by allosterically activating PP2A, which then dephosphorylates ChREBP, allowing the transcription factor to turn on the expression of genes for enzymes of glycolysis and fat synthesis (Fig. 14-28). SUMMARY 14.5 Coordinated Regulation of Glycolysis and Gluconeogenesis Glycolysis and gluconeogenesis are reciprocally regulated to prevent wasteful operation of both pathways at the same time. Hexokinase IV (glucokinase) has kinetic properties related to its special role in the liver: releasing glucose to the blood when blood [glucose] is low; taking up and metabolizing glucose when blood [glucose] is high. Hexokinases I, II, and III are all inhibited by their product, glucose 6-phosphate. PFK-1 is allosterically inhibited by high [ATP]; low [AMP] inhibits FBPase-1. High [ATP] therefore slows glycolysis and speeds gluconeogenesis. Reciprocal allosteric control of glycolysis and gluconeogenesis is mainly achieved by the opposing effects of fructose 2,6- bisphosphate on PFK-1 and FBPase-1. Fructose 2,6-bisphosphate formation is stimulated, indirectly, by insulin, and inhibited by epinephrine. Xylulose 5-phosphate, an intermediate of the pentose phosphate pathway, activates phosphoprotein phosphatase PP2A. Activated PP2A tips the balance toward glucose uptake, glycogen synthesis, and lipid synthesis in the liver. Pyruvate kinase is allosterically inhibited by ATP, and the liver isozyme also is inhibited by cAMP-dependent phosphorylation. When [ATP] is high, glycolysis is slowed. When fatty acids are readily available as fuels, their breakdown in liver mitochondria yields acetyl-CoA, a signal that further oxidation of glucose for fuel is not necessary. Acetyl-CoA activates pyruvate carboxylase, thus favoring gluconeogenesis. Transcription factors such as ChREBP act in the nucleus to regulate the expression of specific genes coding for enzymes of the glycolytic and gluconeogenic pathways. 14.6 Pentose Phosphate Pathway of Glucose Oxidation In most animal tissues, the major catabolic fate of glucose 6-phosphate is glycolytic breakdown to pyruvate, much of which is then oxidized via the citric acid cycle, ultimately leading to the formation of ATP. Glucose 6-phosphate does have other catabolic fates, however, which lead to specialized products needed by the cell. Of particular importance in some tissues is the oxidation of glucose 6-phosphate to pentose phosphates by the pentose phosphate pathway (also called the phosphogluconate pathway or the hexose monophosphate pathway; Fig. 14-29). In this oxidative pathway, NAD P+ is the electron acceptor, yielding NADPH. Rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa, and those of tumors, use the pentose ribose 5-phosphate to make RNA, DNA, and such coenzymes as ATP, NADH, FAD H2, and coenzyme A. FIGURE 14-29 General scheme of the pentose phosphate pathway. NADPH formed in the oxidative phase is used to reduce glutathione, GSSG (see Box 14-4), and to support reductive biosynthesis. The other product of the oxidative phase is ribose 5-phosphate, which serves as a precursor for nucleotides, coenzymes, and nucleic acids. In cells that are not using ribose 5-phosphate for biosynthesis, the nonoxidative phase recycles six molecules of the pentose into five molecules of the hexose glucose 6- phosphate, allowing continued production of NADPH and converting glucose 6-phosphate (in six cycles) to CO2. In other tissues, the essential product of the pentose phosphate pathway is not the pentoses but the electron donor NADPH, needed for reductive biosynthesis or to counter the damaging effects of oxygen radicals. Tissues that carry out extensive fatty acid synthesis (liver, adipose, lactating mammary gland) or very active synthesis of cholesterol and steroid hormones (liver, adrenal glands, gonads) require the NADPH provided by this pathway. Erythrocytes and the cells of the lens and cornea are directly exposed to oxygen and thus to the damaging free radicals generated by oxygen. By maintaining a reducing environment (a high ratio of NADPH to NAD P+ and a high ratio of reduced glutathione to oxidized glutathione), such cells can prevent or undo oxidative damage to proteins, lipids, and other sensitive molecules. In erythrocytes, the NADPH produced by the pentose phosphate pathway is so important in preventing oxidative damage that a genetic defect in glucose 6-phosphate dehydrogenase, the first enzyme of the pathway, can have serious medical consequences (Box 14-4). BOX 14-4 MEDICINE Why Pythagoras Wouldn’t Eat Falafel: Glucose 6- Phosphate Dehydrogenase Deficiency Fava beans, an ingredient of falafel, have been an important food source in the Mediterranean and the Middle East since antiquity. The Greek philosopher and mathematician Pythagoras prohibited his followers from dining on fava beans, perhaps because they make many people sick with a condition called favism, which can be fatal. In favism, erythrocytes begin to lyse 24 to 48 hours a er ingestion of the beans, releasing free hemoglobin into the blood. Jaundice and sometimes kidney failure can result. Similar symptoms can occur with ingestion of the antimalarial drug primaquine or of sulfa antibiotics, or following exposure to certain herbicides. These symptoms have a genetic basis: glucose 6-phosphate dehydrogenase (G6PD) deficiency, which affects about 400 million people worldwide. Most G6PD-deficient individuals are asymptomatic; only the combination of G6PD deficiency and certain environmental factors produces the clinical manifestations. Glucose 6-phosphate dehydrogenase catalyzes the first step in the pentose phosphate pathway (see Fig. 14-30), which produces NADPH. This reductant, essential in many biosynthetic pathways, also protects cells from oxidative damage by hydrogen peroxide (H2O2) and superoxide free radicals, highly reactive oxidants generated as metabolic byproducts and through the actions of drugs such as primaquine and natural products such as divicine — the toxic ingredient of fava beans. During normal detoxification, H2O2 is converted to H2O by reduced glutathione and glutathione peroxidase, and the oxidized glutathione is converted back to the reduced form by glutathione reductase and NADPH (Fig. 1). H2O2 is also broken down to H2O and O2 by catalase, which also requires NADPH. In G6PD-deficient individuals, the NADPH production is diminished and detoxification of H2O2 is inhibited. Cellular damage results: lipid peroxidation leading to breakdown of erythrocyte membranes and oxidation of proteins and DNA. FIGURE 1 Role of NADPH and glutathione in protecting cells against highly reactive oxygen derivatives. Reduced glutathione (GSH) protects the cell by destroying hydrogen peroxide and hydroxyl free radicals. Regeneration of GSH from its oxidized form (GSSG) requires the NADPH produced in the glucose 6-phosphate dehydrogenase reaction. The geographic distribution of G6PD deficiency is instructive. Frequencies as high as 25% occur in tropical Africa, parts of the Middle East, and Southeast Asia, areas where malaria is most prevalent. In addition to such epidemiological observations, in vitro studies show that growth of one malaria parasite, Plasmodium falciparum, is inhibited in G6PD-deficient erythrocytes. The parasite is very sensitive to oxidative damage and is killed by a level of oxidative stress that is tolerable to a G6PD-deficient human host. Because the advantage of resistance to malaria balances the disadvantage of lowered resistance to oxidative damage, natural selection sustains the G6PD-deficient genotype in human populations where malaria is prevalent. Only under overwhelming oxidative stress, caused by drugs, herbicides, or divicine, does G6PD deficiency cause serious medical problems. An antimalarial drug such as primaquine is believed to act by causing oxidative stress to the parasite. It is ironic that antimalarial drugs can cause human illness through the same biochemical mechanism that provides resistance to malaria. Divicine also acts as an antimalarial drug, and ingestion of fava beans may protect against malaria. By refusing to eat falafel, many Pythagoreans with normal G6PD activity may have unwittingly increased their risk of malaria! The Oxidative Phase Produces NADPH and Pentose Phosphates The first reaction of the pentose phosphate pathway (Fig. 14-30) is the oxidation of glucose 6-phosphate by glucose 6- phosphate dehydrogenase (G6PD) to form 6-phosphoglucono-δ - lactone, an intramolecular ester. NAD P+ is the electron acceptor, and the overall equilibrium lies far in the direction of NADPH formation. The lactone is hydrolyzed to the free acid 6- phosphogluconate by a specific lactonase, then 6- phosphogluconate undergoes oxidation and decarboxylation by 6- phosphogluconate dehydrogenase to form the ketopentose ribulose 5-phosphate; the reaction also generates a second molecule of NADPH. Phosphopentose isomerase converts ribulose 5-phosphate to its aldose isomer, ribose 5-phosphate. In some tissues, the pentose phosphate pathway ends at this point, and its overall equation is Glucose6-phosphate+ 2NAD P+ + H2O → ribose 5-phosphate+ C FIGURE 14-30 Oxidative reactions of the pentose phosphate pathway. The end products are ribose 5-phosphate, CO2, and NADPH. The net result is the production of NADPH, a reductant for biosynthetic reactions, and ribose 5-phosphate, a precursor for nucleotide synthesis. The Nonoxidative Phase Recycles Pentose Phosphates to Glucose 6- Phosphate In tissues that require primarily NADPH, the pentose phosphates produced in the oxidative phase of the pathway are recycled into glucose 6-phosphate. In this nonoxidative phase, ribulose 5-phosphate is first epimerized to xylulose 5-phosphate: Then, in a series of rearrangements of the carbon skeletons (Fig. 14-31), six five-carbon sugar phosphates are converted to five six- carbon sugar phosphates, completing the cycle and allowing continued oxidation of glucose 6-phosphate with production of NADPH. Continued recycling leads ultimately to the conversion of glucose 6-phosphate to six CO2. Two enzymes unique to the pentose phosphate pathway act in these interconversions of sugars: transketolase and transaldolase. Transketolase catalyzes the transfer of a two-carbon fragment from a ketose donor to an aldose acceptor (Fig. 14-32a). In its first appearance in the pentose phosphate pathway, transketolase transfers C-1 and C-2 of xylulose 5-phosphate to ribose 5-phosphate, forming the seven- carbon product sedoheptulose 7-phosphate (Fig. 14-32b). The remaining three-carbon fragment from xylulose is glyceraldehyde 3-phosphate. FIGURE 14-31 Nonoxidative reactions of the pentose phosphate pathway. (a) These reactions convert pentose phosphates to hexose phosphates, allowing the oxidative reactions to continue. Transketolase and transaldolase are specific to this pathway; the other enzymes also serve in the glycolytic or gluconeogenic pathways. (b) A schematic diagram showing the pathway from six pentoses (5C) to five hexoses (6C). Note that this involves two sets of the interconversions shown in (a). Every reaction shown here is reversible; unidirectional arrows are used only to make clear the direction of the reactions during continuous oxidation of glucose 6-phosphate. In the light-independent reactions of photosynthesis, the direction of these reactions is reversed. FIGURE 14-32 The first reaction catalyzed by transketolase. (a) The general reaction catalyzed by transketolase is the transfer of a two-carbon group, carried temporarily on enzyme-bound TPP, from a ketose donor to an aldose acceptor. (b) Conversion of two pentose phosphates to a triose phosphate and a seven-carbon sugar phosphate, sedoheptulose 7- phosphate. Next, transaldolase catalyzes a reaction similar to the aldolase reaction of glycolysis: a three-carbon fragment is removed from sedoheptulose 7-phosphate and condensed with glyceraldehyde 3- phosphate, forming fructose 6-phosphate and the tetrose erythrose 4-phosphate (Fig. 14-33). Now transketolase acts again, forming fructose 6-phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate (Fig. 14- 34). Two molecules of glyceraldehyde 3-phosphate formed by two iterations of these reactions can be converted to a molecule of fructose 1,6-bisphosphate as in gluconeogenesis (Fig. 14-16), and finally FBPase-1 and phosphohexose isomerase convert fructose 1,6-bisphosphate to glucose 6-phosphate. Overall, six pentose phosphates have been converted to five hexose phosphates (Fig. 14-32b) — the cycle is now complete. FIGURE 14-33 The reaction catalyzed by transaldolase. FIGURE 14-34 The second reaction catalyzed by transketolase. Transketolase requires the cofactor thiamine pyrophosphate (TPP), which stabilizes a two-carbon carbanion in this reaction (Fig. 14-35a), just as it does in the pyruvate decarboxylase reaction (Fig. 14-13). Transaldolase uses a Lys side chain to form a Schiff base with the carbonyl group of its substrate, a ketose, thereby stabilizing a carbanion (Fig. 14-35b) that is central to the reaction mechanism. FIGURE 14-35 Carbanion intermediates stabilized by covalent interactions with transketolase and transaldolase. (a) The ring of TPP stabilizes the carbanion in the dihydroxyethyl group carried by transketolase. (b) In the transaldolase reaction, the protonated Schiff base formed between the ε -amino group of a Lys side chain and the substrate stabilizes the C-3 carbanion formed a er aldol cleavage. The first and third steps of the oxidative pentose phosphate pathway shown in Figure 14-30 are oxidations with large, negative standard free-energy changes and are essentially irreversible in the cell. The reactions of the nonoxidative part of the pentose phosphate pathway (Fig. 14-31) are readily reversible and thus also provide a means of converting hexose phosphates to pentose phosphates. As we shall see in Chapter 20, a process that converts hexose phosphates to pentose phosphates is central to the photosynthetic assimilation of CO2 by plants. That pathway, the reductive pentose phosphate pathway, is essentially the reversal of the reactions shown in Figure 14-31 and employs many of the same enzymes. All the enzymes of the pentose phosphate pathway are located in the cytosol, like those of glycolysis and most of those of gluconeogenesis. In fact, these three pathways are connected through several shared intermediates and enzymes. The glyceraldehyde 3-phosphate formed by the action of transketolase is readily converted to dihydroxyacetone phosphate by the glycolytic enzyme triose phosphate isomerase, and these two trioses can be joined by the aldolase as in gluconeogenesis, forming fructose 1,6-bisphosphate. Alternatively, the triose phosphates can be oxidized to pyruvate by the glycolytic reactions. The fate of the trioses is determined by the cell’s relative needs for pentose phosphates, NADPH, and ATP. Glucose 6-Phosphate Is Partitioned between Glycolysis and the Pentose Phosphate Pathway Whether glucose 6-phosphate enters glycolysis or the pentose phosphate pathway depends on the current needs of the cell and on the concentration of NAD P+ in the cytosol. Without this electron acceptor, the first reaction of the pentose phosphate pathway (catalyzed by glucose 6-phosphate dehydrogenase) cannot proceed. When a cell is rapidly converting NADPH to NAD P+ in biosynthetic reductions, [NAD P+] rises, allosterically stimulating glucose 6-phosphate dehydrogenase and thereby increasing the flux of glucose 6-phosphate through the pentose phosphate pathway (Fig. 14-36). When the demand for NADPH slows, the level of NAD P+ drops, the pentose phosphate pathway slows, and glucose 6-phosphate is instead used to fuel glycolysis. FIGURE 14-36 Role of NADPH in regulating the partitioning of glucose 6-phosphate between glycolysis and the pentose phosphate pathway. When NADPH is forming faster than it is being used for biosynthesis and glutathione reduction, [NADPH] rises and inhibits the first enzyme in the pentose phosphate pathway. As a result, more glucose 6-phosphate is available for glycolysis. Thiamine Deficiency Causes Beriberi and Wernicke-Korsakoff Syndrome Thiamine, precursor to the cofactor thiamine pyrophosphate (TPP), is one of the B vitamins, essential in humans. Lack of vitamin B1 in the diet leads to a range of medical problems. The condition known as beriberi is characterized by an accumulation of body fluids (swelling), pain, paralysis, and ultimately, without treatment, death. Wernicke-Korsakoff syndrome, also caused by a severe deficiency of thiamine, typically includes problems with voluntary movements, reflected in abnormal eye movements and gait, and neurological defects. The syndrome is more common among heavy drinkers than in the general population because chronic, heavy alcohol consumption interferes with the intestinal absorption of thiamine. The syndrome can be exacerbated by a mutation in the gene for transketolase that results in an enzyme with a lowered affinity for TPP — an affinity one-tenth that of the normal enzyme. This defect makes individuals much more sensitive to a thiamine deficiency: even a moderate thiamine deficiency (tolerable in individuals with an unmutated transketolase) can result in a transketolase that is not saturated with TPP at its normal concentration. The result is a slowing down of the whole pentose phosphate pathway. In people with Wernicke-Korsakoff syndrome, this mutation results in a worsening of symptoms, which can include severe memory loss, mental confusion, and partial paralysis.

SUMMARY 14.6 Pentose Phosphate Pathway of Glucose Oxidation The oxidative pentose phosphate pathway produces NADPH and pentose phosphates. Tissues that carry out extensive fatty acid synthesis (liver, adipose, lactating mammary gland) or very active synthesis of cholesterol and steroid hormones (liver, adrenal glands, gonads) require the NADPH provided by this pathway. Ribose 5-phosphate is a precursor for nucleotide and nucleic acid synthesis. The first, oxidative phase of the pentose phosphate pathway consists of two oxidations that convert glucose 6-phosphate to ribulose 5-phosphate and reduce NAD P+ to NADPH. The second, nonoxidative phase of the pentose phosphate pathway comprises steps that convert pentose phosphates to glucose 6-phosphate, which begins the oxidative cycle again. Entry of glucose 6-phosphate either into glycolysis or into the pentose phosphate pathway is largely determined by the relative concentrations of NAD P+ and NADPH. Chapter Review KEY TERMS Terms in bold are defined in the glossary. glycolysis hexokinase isozymes phosphofructokinase-1 (PFK-1) fructose 1,6-bisphosphate aldolase aldolase triose phosphate isomerase glyceraldehyde 3-phosphate dehydrogenase acyl phosphate phosphoglycerate kinase substrate-level phosphorylation respiration-linked phosphorylation phosphoglycerate mutase enolase phosphoenolpyruvate (PEP) pyruvate kinase glycogen phosphorylase mutases isomerases lactose intolerance galactosemia hypoxia fermentation lactic acid fermentation ethanol (alcohol) fermentation lactate dehydrogenase pyruvate decarboxylase alcohol dehydrogenase thiamine pyrophosphate (TPP) gluconeogenesis biotin fructose 1,6-bisphosphatase (FBPase-1) glucogenic glyceroneogenesis glucagon fructose 2,6-bisphosphate phosphofructokinase-2 (PFK-2) fructose 2,6-bisphosphatase (FBPase-2) carbohydrate response element binding protein (ChREBP) pentose phosphate pathway phosphogluconate pathway hexose monophosphate pathway glucose 6-phosphate dehydrogenase (G6PD) 6-phosphogluconate dehydrogenase PROBLEMS 1. Is the Hexokinase Reaction at Equilibrium in Cells? For the reaction catalyzed by the enzyme hexokinase Glucose+ AT P ⇌ glucose6-phosphate+ AD P Glucose+ AT P ⇌ glucose6-phosphate+ AD P the equilibrium constant, Keq, is 7.8× 102. In living E. coli cells, [AT P]= 5m M , [AD P]= 0.5m M , [glucose]= 2m M , and [glucose6-phosphate]= 1m M . Is the reaction at equilibrium in E. coli? 2. Equation for the Preparatory Phase of Glycolysis Write balanced biochemical equations for all the reactions in the catabolism of glucose to two molecules of glyceraldehyde 3- phosphate (the preparatory phase of glycolysis), including the standard free-energy change for each reaction. Then write the overall or net equation for the preparatory phase of glycolysis, with the net standard free-energy change. 3. Payoff Phase of Glycolysis: Fate of Pyruvate in Active Skeletal Muscle In working skeletal muscle under anaerobic conditions, glyceraldehyde 3-phosphate is converted to pyruvate (the payoff phase of glycolysis), and the pyruvate is reduced to lactate. Write balanced biochemical equations for all the reactions in this process, with the standard free- energy change for each reaction. Then write the overall or net equation for the payoff phase of glycolysis with fermentation to lactate, including the net standard free- energy change. 4. Energetics of the Aldolase Reaction Aldolase catalyzes the glycolytic reaction Fructose1,6-bisphosphate→ glyceraldehyde Fructose1,6bisphosphate→ glyceraldehyde 3-phosphate+ dihydroxyacetonephosphate The standard free-energy change for this reaction in the direction written is +23.8kJ /mol. The concentrations of the three intermediates in the hepatocyte of a mammal are fructose 1,6-bisphosphate, 1.4× 10−5M ; glyceraldehyde 3- phosphate, 3× 10−6M ; and dihydroxyacetone phosphate, 1.6× 10−5M . At body temperature (37 °C), what is the actual free-energy change for the reaction? 5. Equivalence of Triose Phosphates A researcher adds 14C- labeled glyceraldehyde 3-phosphate to a yeast extract. Aer a short time, she isolates fructose 1,6-bisphosphate labeled with 14C at C-3 and C-4. What was the location of the 14C label in the starting glyceraldehyde 3-phosphate? Where did the second 14C label in fructose 1,6-bisphosphate come from? Explain. 6. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction Would shortening the glycolytic pathway in this way benefit the cell? Explain. 7. Role of Lactate Dehydrogenase During strenuous activity, the demand for ATP in muscle tissue vastly increases. In rabbit leg muscle or turkey flight muscle, ATP production is almost exclusively a product of lactic acid fermentation. Phosphoglycerate kinase and pyruvate kinase catalyze the two reactions that form ATP in the payoff phase of glycolysis. Suppose skeletal muscle were devoid of lactate dehydrogenase. Could it carry out strenuous physical activity; that is, could it generate ATP at a high rate by glycolysis? Explain. 8. Efficiency of ATP Production in Muscle The transformation of glucose to lactate in myocytes releases only about 7% of the free energy released when glucose is completely oxidized to CO2 and H2O. Does this mean that glycolysis with lactate fermentation under anaerobic conditions in muscle is a wasteful use of glucose? Explain. 9. Free-Energy Change for Triose Phosphate Oxidation The oxidation of glyceraldehyde 3-phosphate to 1,3- bisphosphoglycerate, catalyzed by glyceraldehyde 3- phosphate dehydrogenase, proceeds with an unfavorable equilibrium constant (K′eq = 0.08; ΔG′°= 6.3kJ /mol), yet the flow through this point in the glycolytic pathway proceeds smoothly. How does the cell overcome the unfavorable equilibrium? 10. Arsenate Poisoning Arsenate is structurally and chemically similar to inorganic phosphate (Pi), and many enzymes that require phosphate will also use arsenate. Organic compounds of arsenate are less stable than analogous phosphate compounds, however. For example, acyl arsenates decompose rapidly by hydrolysis, as shown. On the other hand, acyl phosphates, such as 1,3- bisphosphoglycerate, are more stable and undergo further enzyme-catalyzed transformation in cells. a. Predict the effect on the net reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase if phosphate were replaced by arsenate. b. What would be the consequence to an organism if arsenate were substituted for phosphate? Arsenate is very toxic to most organisms. Explain why. 11. Requirement for Phosphate in Ethanol Fermentation In 1906 Harden and Young, in a series of classic studies on the fermentation of glucose to ethanol and CO2 by extracts of brewer’s yeast, made the following observations: (1) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used. (2) During fermentation under these conditions (with no phosphate), ethanol, CO2, and a hexose bisphosphate accumulated. (3) When arsenate was substituted for phosphate, no hexose bisphosphate accumulated, but the fermentation proceeded until all the glucose was converted to ethanol and CO2. a. Why did fermentation cease when the supply of phosphate was exhausted? b. Why did ethanol and CO2 accumulate? Was the conversion of pyruvate to ethanol and CO2 essential? Why? Identify the hexose bisphosphate that accumulated. Why did it accumulate? c. Why did the substitution of arsenate for phosphate prevent the accumulation of the hexose bisphosphate yet allow fermentation to ethanol and CO2 to go to completion? (See Problem 10.) 12. Role of the Vitamin Niacin Adults engaged in strenuous physical activity require an intake of about 160 g of carbohydrate daily but only about 20 mg of niacin for optimal nutrition. Given the role of niacin in glycolysis, how do you explain the observation? 13. Synthesis of Glycerol Phosphate The glycerol 3- phosphate required for the synthesis of glycerophospholipids can be synthesized from a glycolytic intermediate. Propose a reaction sequence for this conversion. 14. Severity of Clinical Symptoms Due to Enzyme Deficiency The clinical symptoms of two forms of galactosemia — galactokinase-deficiency galactosemia and transferase-deficiency galactosemia — show radically different severity. Although both types produce gastric discomfort aer milk ingestion, deficiency of the transferase also leads to liver, kidney, spleen, and brain dysfunction and eventual death. What products accumulate in the blood and tissues with each type of enzyme deficiency? Estimate the relative toxicities of these products from the above information. 15. Ethanol Affects Blood Glucose Levels The consumption of alcohol (ethanol), especially aer periods of strenuous activity or aer not eating for several hours, results in a deficiency of glucose in the blood, a condition known as hypoglycemia. The first step in the metabolism of ethanol by the liver is oxidation to acetaldehyde, catalyzed by liver alcohol dehydrogenase: CH3CH2OH + NAD + → CH3CHO + NAD H + H+ Explain how this reaction inhibits the transformation of lactate to pyruvate. Why does this lead to hypoglycemia? 16. Blood Lactate Levels during Vigorous Exercise The graph shows the concentrations of lactate in blood plasma before, during, and aer a 400 m sprint. a. What causes the rapid rise in lactate concentration? b. What causes the decline in lactate concentration aer completion of the sprint? Why does the decline occur more slowly than the increase? c. Why is the concentration of lactate not zero during the resting state? 17. Relationship between Fructose 1,6-Bisphosphatase and Blood Lactate Levels A congenital defect in the liver enzyme fructose 1,6-bisphosphatase results in abnormally high levels of lactate in the blood plasma. Explain. 18. Effect of O2 Supply on Glycolytic Rates The regulated steps of glycolysis in intact cells can be identified by studying the catabolism of glucose in whole tissues or organs. For example, the glucose consumption by heart muscle can be measured by artificially circulating blood through an isolated intact heart and measuring the concentration of glucose before and aer the blood passes through the heart. If the circulating blood is deoxygenated, heart muscle consumes glucose at a steady rate. When oxygen is added to the blood, the rate of glucose consumption drops dramatically, then is maintained at the new, lower rate. Explain. 19. Regulation of PFK-1 The graph shows the effect of ATP on the allosteric enzyme PFK-1. For a given concentration of fructose 6-phosphate, the PFK-1 activity increases with increasing concentrations of ATP, but there is a point beyond which increasing the concentration of ATP inhibits the enzyme. a. Explain how ATP can be both a substrate and an inhibitor of PFK-1. How is the enzyme regulated by ATP? b. How do ATP levels regulate glycolysis? c. The inhibition of PFK-1 by ATP diminishes when the ADP concentration is high, as shown in the graph. What explains this observation? 20. Cellular Glucose Concentration Homeostatic mechanisms maintain the concentration of glucose in human blood at about 5 mM. The concentration of free glucose inside a myocyte is much lower. Why is the concentration so low in the cell? What happens to glucose aer entry into the cell? Physicians administer glucose intravenously as a food source in certain clinical situations. Given that the transformation of glucose to glucose 6- phosphate consumes ATP, why not administer intravenous glucose 6-phosphate instead? 21. Ethanol Production in Yeast When grown anaerobically on glucose, yeast (S. cerevisiae) converts pyruvate to acetaldehyde, then reduces acetaldehyde to ethanol using electrons from NADH. Write the equation for the second reaction, and calculate its equilibrium constant at 25 °C, given the standard reduction potentials in Table 13-7. 22. Pathway of Atoms in Fermentation An investigator carries out a “pulse-chase” experiment using 14C-labeled carbon sources on a yeast extract maintained under strictly anaerobic conditions to produce ethanol. The experiment consists of incubating a small amount of 14C-labeled substrate (the pulse) with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The addition of excess unlabeled glucose then “chases” the label through the pathway. The chase effectively prevents any further entry of labeled glucose into the pathway. a. If the investigator uses [1-14C]glucose (glucose labeled at C-1 with 14C) as a substrate, what is the location of 14C in the product ethanol? Explain. b. Where would 14C have to be located in the starting glucose to ensure that all the 14C activity is liberated as 14CO2 during fermentation to ethanol? Explain. 23. Heat from Fermentations Large-scale industrial fermenters generally require constant, vigorous cooling. Why? 24. Fermentation to Produce Soy Sauce Soy sauce preparation involves fermenting a salted mixture of soybeans and wheat with several microorganisms, including yeast, over a period of 8 to 12 months. The resulting sauce (aer solids are removed) is rich in lactate and ethanol. How are these two compounds produced? To prevent the soy sauce from having a strong vinegary taste (vinegar is dilute acetic acid), oxygen must be kept out of the fermentation tank. Why? 25. Glucogenic Substrates A common procedure for determining the effectiveness of compounds as precursors of glucose in mammals is to starve the animal until the liver glycogen stores are depleted and then administer the compound in question. A substrate that leads to a net increase in liver glycogen is termed glucogenic, because it must first be converted to glucose 6-phosphate. Show by means of known enzymatic reactions which of these substances are glucogenic: 26. Pathway of Atoms in Gluconeogenesis An investigator briefly incubates a liver extract capable of carrying out all the normal metabolic reactions of the liver in separate experiments with two different 14C-labeled precursors: [14C]bicarbonate and [14C]pyruvate. Trace the pathway of each precursor through gluconeogenesis. Indicate the location of 14C in all intermediates and in the product, glucose. 27. Energy Cost of a Cycle of Glycolysis and Gluconeogenesis What is the cost (in ATP equivalents) of transforming glucose to pyruvate via glycolysis and back again to glucose via gluconeogenesis? 28. Relationship between Gluconeogenesis and Glycolysis Why is it important that gluconeogenesis is not the exact reversal of glycolysis? 29. Energetics of the Pyruvate Kinase Reaction Explain in bioenergetic terms how the conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis overcomes the large, negative, standard free-energy change of the pyruvate kinase reaction in glycolysis. 30. Muscle Wasting in Starvation One consequence of starvation is a reduction in muscle mass. What happens to the muscle proteins? 31. Effect of Phloridzin on Carbohydrate Metabolism Phloridzin, a toxic glycoside from the bark of the pear tree, blocks the normal reabsorption of glucose from the kidney tubule, thus causing blood glucose to be almost completely excreted in the urine. In an experiment, rats fed phloridzin and sodium succinate excreted about 0.5 mol of glucose (made by gluconeogenesis) for every 1 mol of sodium succinate ingested. How do rats transform the succinate to glucose? Explain the stoichiometry. 32. Excess O2 Uptake during Gluconeogenesis The conversion of lactate to glucose in the liver requires the input of 6 mol of ATP for every mol of glucose produced. Investigators can monitor the extent of this process in a rat liver preparation by administering [14C]lactate and measuring the amount of [14C]glucose produced. Because the stoichiometry of O2 consumption and ATP production is known (about 5 ATP per O2), investigators can predict the extra O2 consumption above the normal rate aer administering a given amount of lactate. However, when they actually measure extra O2 used in the synthesis of glucose from lactate, it is always higher than what the stoichiometric relationships predict. Suggest a possible explanation for this observation. 33. Role of the Pentose Phosphate Pathway If the oxidation of glucose 6-phosphate via the pentose phosphate pathway were being used primarily to generate NADPH for biosynthesis, the other product, ribose 5-phosphate, would accumulate. What problems might this cause? DATA ANALYSIS PROBLEM 34. Engineering a Fermentation System Fermentation of plant matter to produce ethanol for fuel is one potential method for reducing the use of fossil fuels and thus the CO2 emissions that lead to global warming. Many microorganisms can break down cellulose, then ferment the glucose to ethanol. However, many potential cellulose sources, including agricultural residues and switchgrass, also contain substantial amounts of arabinose, which is not as easily fermented. Escherichia coli is capable of fermenting arabinose to ethanol, but it is not naturally tolerant of high ethanol levels, thus limiting its utility for commercial ethanol production. Another bacterium, Zymomonas mobilis, is naturally tolerant of high levels of ethanol but cannot ferment arabinose. Deanda, Zhang, Eddy, and Picataggio (1996) described their efforts to combine the most useful features of these two organisms by introducing the E. coli genes for the arabinose- metabolizing enzymes into Z. mobilis. a. Why is this a simpler strategy than the reverse: engineering E. coli to be more ethanol-tolerant? Deanda and colleagues inserted five E. coli genes into the Z. mobilis genome: araA, coding for L-arabinose isomerase, which interconverts L-arabinose and L- ribulose; araB, L-ribulokinase, which uses ATP to phosphorylate L-ribulose at C-5; araD, L-ribulose 5- phosphate epimerase, which interconverts L-ribulose 5-phosphate and L-xylulose 5-phosphate; talB, transaldolase; and tktA, transketolase. b. For each of the three ara enzymes, briefly describe the chemical transformation it catalyzes and, where possible, name an enzyme discussed in this chapter that carries out an analogous reaction. The five E. coli genes inserted in Z. mobilis allowed the entry of arabinose into the nonoxidative phase of the pentose phosphate pathway (Fig. 14-31), where it was converted to glucose 6-phosphate and fermented to ethanol. c. The three ara enzymes eventually converted arabinose into which sugar? d. The product from part (c) feeds into the pathway shown in Figure 14-31a. Combining the five E. coli enzymes listed above with the enzymes of this pathway, describe the overall pathway for the fermentation of six molecules of arabinose to ethanol. e. What is the stoichiometry of the fermentation of six molecules of arabinose to ethanol and CO2? How many ATP molecules would you expect this reaction to generate? f. Z. mobilis uses a pathway for ethanol fermentation that is slightly different from the one described in this chapter. As a result, the expected ATP yield is only 1 ATP per molecule of arabinose. Although this is less beneficial for the bacterium, it is better for ethanol production. Why? Another sugar commonly found in plant matter is xylose. g. What additional enzymes would you need to introduce into the modified Z. mobilis strain described above to enable it to use xylose as well as arabinose to produce ethanol? You don’t need to name the enzymes (they may not even exist in the real world); just give the reactions they would need to catalyze. Reference Deanda, K., M. Zhang, C. Eddy, and S. Picataggio. 1996. Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl. Environ. Microbiol. 62:4465–4470.