Glucose uptake into the cells is regulated

Glucose is not sufficiently lipid soluble to enter cells by passive diffusion across the plasma membrane. Dietary glucose enters the intestinal mucosal cells mainly by sodium cotransport, but the uptake of glucose from blood or interstitial fluid into cells occurs by facilitated diffusion.

Table 21.1 summarizes the most important facilitated-diffusion glucose carriers. One of these carriers, GLUT4, is insulin dependent. Its deposition in the plasma membrane is enhanced by insulin (Fig. 21.3). The consequence is that muscle and adipose tissue take up glucose after a carbohydrate-rich meal, when the insulin level is high, but not during fasting, when the insulin level is low. Muscle and adipose tissue do not depend on glucose but can subsist on fatty acids and other nutrients if needed. During fasting, glucose is redirected from muscle and adipose tissue to tissues that depend on glucose, including brain and erythrocytes.

Table 21.1 Most Important Glucose Transporters

Figure 21.3 Effect of insulin on the glucose carriers in adipocytes. A similar mechanism is thought to operate in skeletal muscle. A, In the resting state, most glucose carriers are present in the membrane of intracellular vesicles. B, After insulin binding and receptor autophosphorylation, the carrier-containing vesicles fuse with the plasma membrane. This leads to an increased V_max of glucose transport across the plasma membrane.

Glucose degradation begins in the cytoplasm and ends in the mitochondria

The steps in glucose oxidation are summarized in Figure 21.4. The initial reaction sequence, known as glycolysis, is cytoplasmic. It turns one molecule of glucose (six carbons) into two molecules of the three-carbon compound pyruvate. All cells of the body are capable of glycolysis.

Figure 21.4 Steps in glucose oxidation. The catabolic pathways convert the carbon of the substrate to carbon dioxide. The hydrogen initially is transferred to the coenzymes NAD⁺ and FAD. The reduced coenzymes then are reoxidized by the respiratory chain. Most of the ATP is produced by oxidative phosphorylation, which couples the oxidation of the reduced coenzymes to ATP synthesis.

Under aerobic conditions, pyruvate is transported into the mitochondrion, where it is turned into the two-carbon compound acetyl-CoA. Acetyl-CoA then enters the TCA cycle by reacting with the four-carbon compound oxaloacetate to form the six-carbon compound citrate. Citrate is converted back to oxaloacetate in the remaining reactions of the TCA cycle.

In these pathways, the hydrogen of the substrate is transferred to the coenzymes NAD⁺ and FAD (see Chapter 5). The reduced coenzymes donate electrons to the respiratory chain of the inner mitochondrial membrane, which in turn donates them to molecular oxygen. The reoxidation of the reduced coenzymes is highly exergonic. It is the energy source for ATP synthesis by oxidative phosphorylation. The TCA cycle and oxidative phosphorylation take place in all cells that contain mitochondria.

Glycolysis begins with atp-dependent phosphorylations

After entering the cell, glucose is phosphorylated to glucose-6-phosphate by hexokinase (Fig. 21.5). The hexokinase reaction is irreversible for two reasons: Its ΔG^0′ is strongly negative (−4.0 kcal/mol) because an energy-rich phosphoanhydride bond in ATP is cleaved while a “low-energy” phosphate ester bond is formed (Table 21.2), and the ATP concentration in a healthy cell is always far higher than the ADP concentration.

Figure 21.5 Reactions of glycolysis, the major catabolic pathway for glucose. It is active in the cytoplasm of all cells in the human body.

Table 21.2 Standard Free Energy Changes of Glycolytic Reactions

Most glycolytic intermediates have three carbons

The six-carbon intermediate fructose-1,6-bisphosphate is cleaved into two triose phosphates by the enzyme aldolase. Carbons 1, 2, and 3 of the sugar form dihydroxyacetone phosphate, and carbons 4, 5, and 6 form glyceraldehyde-3-phosphate. The triose phosphates are interconverted in the reversible triose phosphate isomerase reaction.

Aldolase and triose phosphate isomerase establish an equilibrium between fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde-3-phosphate. Although only glyceraldehyde-3-phosphate proceeds through the remaining glycolytic reactions, triose phosphate isomerase ensures that all six glucose-derived carbons can proceed through the pathway.

Glyceraldehyde-3-phosphate is converted to the energy-rich intermediate 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase. The enzyme couples the exergonic oxidation of the aldehyde group in the substrate with the endergonic formation of an energy-rich bond between the newly created carboxyl group and inorganic phosphate. The reaction also forms a substrate for oxidative phosphorylation by reducing NAD⁺ to NADH.

Simple hydrolysis of the mixed anhydride bond in 1,3-bisphosphoglycerate would release 11.8 kcal/mol in the form of heat. Rather than wasting this energy by hydrolyzing the bond, the enzyme phosphoglycerate kinase transfers the phosphate to ADP, forming ATP. This strategy of forming an energy-rich intermediate that is then used for ATP synthesis is called substrate-level phosphorylation.

3-Phosphoglycerate is isomerized to 2-phosphoglycerate by phosphoglycerate mutase. “Mutase” is an old-fashioned name for isomerases that shift the position of a phosphate group in the molecule. 2-Phosphoglycerate, in turn, is dehydrated to phosphoenolpyruvate (PEP) by enolase.

The last enzyme of glycolysis, pyruvate kinase, makes substrate-level phosphorylation by transferring the phosphate group of PEP to ADP. Although ATP is synthesized, this reaction is highly exergonic with a standard free energy change (ΔG^0′) of −7.5 kcal/mol. This implies a free energy content of 14.8 kcal/mol for the phosphate ester bond in PEP. Why is this phosphate ester so unusually energy rich? The initial transfer of phosphate from PEP to ADP is indeed endergonic. However, the enolpyruvate formed in this reaction rearranges almost immediately to pyruvate. This highly exergonic reaction removes enolpyruvate from the equilibrium (Fig. 21.6).

Figure 21.6 Pyruvate kinase reaction. Pyruvate shows keto-enol tautomerism, the keto form being energetically far more stable than the enol form.

Overall, the reactions of glycolysis produce a net yield of two ATP molecules and two NADH molecules for each molecule of glucose (Table 21.3).

Table 21.3 Products Formed during Conversion of One Molecule of Glucose to Two Molecules of Pyruvate in Aerobic Glycolysis*

* Note that all reactions beyond the aldolase reaction occur twice for each glucose molecule.

Only the hexokinase, PFK, and pyruvate kinase reactions are “irreversible.” The aldolase and triose phosphate isomerase reactions have very unfavorable equilibria (see Table 21.2). Nevertheless they can proceed because fructose-1,6-bisphosphate is formed in the irreversible PFK reaction and glyceraldehyde-3-phosphate is rapidly consumed in the next reactions of the pathway. The actual equilibrium of the glyceraldehyde-3-phosphate dehydrogenase reaction is far more favorable than suggested by its ΔG^0′ value of +1.5 kcal/mol because NAD⁺ is far more abundant than NADH in the aerobic cell.

Phosphofructokinase is the most important regulated enzyme of glycolysis

Most tissues glycolyze heavily after a carbohydrate meal but switch to fatty acid oxidation during fasting. The long-term control of glycolysis, particularly in the liver, is affected by changes in the amounts of some key glycolytic enzymes, triggered by nutrients and hormones. In general, insulin and glucose increase the levels of glycolytic enzymes, whereas glucagon and fatty acids have the opposite effect.

Of the important hormones, insulin rises in response to elevated blood glucose after a meal. It stimulates glucose consumption in many tissues, both by glycolysis and by other pathways. Glucagon rises in response to low blood glucose during fasting. It reduces glucose consumption and stimulates glucose production by the liver.

The short-term control of glycolysis depends mainly on the allosteric enzyme PFK, which catalyzes the committed step of glycolysis. PFK is

The response to adenine nucleotides ensures that glycolytic activity increases when more ATP is needed (e.g., in contracting muscle). Citrate is a mitochondrial metabolite that signals an abundance of energy and metabolic intermediates, and low pH dampens glycolytic activity when pyruvic and lactic acid, the end products of glycolysis, accumulate to dangerous levels.

Additional control sites are insulin-dependent glucose uptake into the cell by the GLUT4 transporter in muscle and adipose tissue as well as the other irreversible enzymes of glycolysis, hexokinase and pyruvate kinase. In most tissues (but not the liver), hexokinase is competitively inhibited by its own product, glucose-6-phosphate. This prevents the accumulation of glucose-6-phosphate when the supply of glucose exceeds the capacity of the metabolizing pathways. Glucose-6-phosphate must not be allowed to accumulate because it would tie up the cell’s phosphate and thereby impair ATP synthesis. Pyruvate kinase, finally, is inhibited by ATP in many tissues, including the liver.

Lactate is produced under anaerobic conditions

Glycolysis produces ATP without consuming oxygen. Does this mean that we can live without oxygen by turning glucose into pyruvate? Not quite. The immediate problem is that glycolysis turns NAD⁺ into NADH. Without a mechanism to regenerate NAD⁺, glycolysis would soon grind to a screeching halt for lack of NAD⁺.

The solution to this problem is simple (Fig. 21.7). The enzyme lactate dehydrogenase (LDH) regenerates NAD⁺ by transferring the hydrogen of NADH to the keto group of pyruvate:

Figure 21.7 Anaerobic glycolysis. Lactate dehydrogenase regenerates NAD⁺ for the glyceraldehyde-3-phosphate dehydrogenase reaction. The conversion of glucose to lactic acid can proceed smoothly with a net synthesis of two ATP molecules.

The equilibrium of the LDH reaction favors lactate, but the reaction is physiologically reversible because NAD⁺ is far more abundant than NADH under aerobic conditions. In fact, lactate is a metabolic dead end. The LDH reaction is the only way to channel lactate back into the metabolic pathways.

The overall balance of lactate formation by anaerobic glycolysis is

Thus it is possible to make ATP in the absence of oxygen. Carbohydrates are the only metabolic substrates that can produce ATP under anaerobic conditions. A major limitation of anaerobic glycolysis is that the protons that are formed along with the lactate anion can create a serious pH problem.

Another limitation is that the two ATP molecules formed in glycolysis capture only 14.6 kcal of useful energy, whereas the complete oxidation of glucose produces approximately 270 kcal (see Table 21.7). Therefore anaerobic glycolysis is useful only under certain circumstances, for example:

Table 21.7 Energy Yield from Glucose Oxidation

⁎ The energy yield from cytoplasmic NADH depends on the shuttle system used.

CLINICAL EXAMPLE 21.3: Lactic Acidosis

The overproduction or underutilization of lactic acid leads to lactic acidosis. The most common cause is impairment of oxidative metabolism by respiratory failure, insufficient oxygen transport, or toxins that prevent oxidative phosphorylation. As in tissue ischemia, PFK is stimulated by low energy charge, and large amounts of lactate are formed by glycolysis. Without oxygen, the mitochondria cannot oxidize NADH to NAD⁺. As a result, the accumulating NADH makes the LDH reaction irreversible in the direction of lactate formation.

Some other causes of lactic acidosis are listed in Table 21.4. In alcohol intoxication, for example, the rapid formation of NADH during alcohol oxidation increases the [NADH]/[NAD⁺] ratio. This makes the liver unable to oxidize lactate, and lactate is released into the blood.

Table 21.4 Conditions Resulting in Lactic Acidosis

Condition	Mechanism
Physical exercise	Anaerobic glycolysis in muscle
Severe lung disease High altitude Drowning	Impaired respiration
Severe anemia Carbon monoxide poisoning Sickling crisis	Impaired oxygen delivery
Cyanide poisoning	Inhibition of oxidative phosphorylation
Alcohol intoxication von Gierke disease	Elevated [NADH]/[NAD+] ratio Impaired gluconeogenesis
Pyruvate dehydrogenase deficiency	Impaired pyruvate oxidation
Leukemia Metastatic carcinoma	Anaerobic glycolysis by neoplastic cells

Pyruvate is decarboxylated to acetyl-CoA in the mitochondria

Under aerobic conditions, pyruvate is oxidized in the mitochondria. It diffuses through the pores in the outer mitochondrial membrane and is transported across the inner mitochondrial membrane into the mitochondrial matrix, where it is oxidatively decarboxylated to acetyl-CoA:

In addition to the tightly bound prosthetic groups, the cosubstrates NAD⁺ and CoA are required for the reaction.

The structures of thiamin pyrophosphate (TPP) and lipoic acid are shown in Figure 21.8. TPP acts as a carrier of pyruvate and of the hydroxyethyl group that is formed by pyruvate decarboxylation. Lipoic acid participates as a redox system and carrier of the acetyl group. The reaction sequence is shown in Figure 21.9.

Figure 21.8 Structures of thiamin pyrophosphate (TPP) and lipoic acid. In the pyruvate dehydrogenase complex, TPP is bound noncovalently to the apoprotein, whereas lipoic acid is bound covalently by an amide bond with a lysine side chain.

Figure 21.9 Pyruvate dehydrogenase reaction.

With the exception of lipoic acid, the coenzymes of pyruvate dehydrogenase require vitamins for their synthesis: pantothenic acid (CoA), niacin (NAD), riboflavin (FAD), and thiamin (TPP). A deficiency of any of these vitamins can impair the pyruvate dehydrogenase reaction. In thiamin deficiency (beriberi), for example, the blood levels of pyruvate, lactate, and alanine are elevated after a carbohydrate-rich meal. Pyruvate accumulates because its major reaction is blocked, and most of it is either reduced to lactate or transaminated to alanine.

The TCA cycle produces two molecules of carbon dioxide for each acetyl residue

The TCA cycle, also known as the citric acid cycle or Krebs cycle, is the final common pathway for the oxidation of all major nutrients. It takes place in the mitochondrial matrix, and it is active in all cells that possess mitochondria.

In the first reaction, the acetyl group of acetyl-CoA reacts with the four-carbon compound oxaloacetate to form the six-carbon compound citrate. This irreversible reaction (Table 21.5) is catalyzed by citrate synthase. The remaining reactions regenerate oxaloacetate from citrate, with two carbons released as carbon dioxide (Fig. 21.12).

Table 21.5 Standard Free Energy Changes (ΔG^0′) of Pyruvate Dehydrogenase Reaction and Tricarboxylic Acid Cycle Reactions

Figure 21.12 Tricarboxylic acid (TCA) cycle.

CLINICAL EXAMPLE 21.4: Arsenic Poisoning

Arsenic occurs in two forms that are toxic by different mechanisms. Arsenate is a structural analog of phosphate that competes with phosphate in many biochemical reactions. However, the bonds that arsenate forms with phosphate and carboxyl groups are unstable and hydrolyze spontaneously. Figure 21.10 shows how it uncouples substrate-level phosphorylation in glycolysis. The term “uncoupling” implies that the pathway can proceed, but without ATP synthesis.

Figure 21.10 Uncoupling of substrate-level phosphorylation in glycolysis by arsenate. An unstable mixed anhydride is formed between arsenate and 3-phosphoglycerate. This anhydride hydrolyzes spontaneously. The pathway can proceed because the product of this hydrolysis, 3-phosphoglycerate, is a normal glycolytic intermediate.

Arsenite is even more toxic than arsenate. It poisons pyruvate dehydrogenase and other lipoic acid containing enzymes by binding to the sulfhydryl groups in dihydrolipoic acid (Fig. 21.11). A similar reaction of arsenite with closely spaced sulfhydryl groups in immature keratin leads to its incorporation in hair and fingernails. Its determination in hair is used forensically in cases of alleged arsenic poisoning.

Figure 21.11 Reaction of arsenite with dihydrolipoic acid.

Citrate is isomerized to isocitrate by aconitase. The enzyme first dehydrates citrate to aconitate and then hydrates aconitate to isocitrate (Fig. 21.13). At equilibrium, the composition is 90% citrate, 3% aconitate, and 7% isocitrate.

Figure 21.13 Aconitase reaction.

Fluoroacetate has occasionally been used as a rat poison but is interesting for terrorists as well. It is metabolically converted to fluorocitrate by the same enzymes that otherwise metabolize acetate (Fig. 21.14). The resulting fluorocitrate is a potent inhibitor of aconitase.

Figure 21.14 Metabolic activation of fluoroacetate to fluorocitrate. Fluorocitrate is an inhibitor of aconitase.

Isocitrate is oxidatively decarboxylated to α-ketoglutarate (2-oxoglutarate) by isocitrate dehydrogenase. Oxalosuccinate is an enzyme-bound intermediate in this reaction (Fig. 21.15). The isocitrate dehydrogenase of the TCA cycle is an NAD-linked enzyme.

Figure 21.15 Isocitrate dehydrogenase reaction.

The next enzyme of the cycle, α-ketoglutarate dehydrogenase, resembles pyruvate dehydrogenase in structure, reaction mechanism, and coenzyme requirements. However, it works on α-ketoglutarate rather than pyruvate and produces succinyl-CoA rather than acetyl-CoA.

In the reversible succinyl-CoA synthetase (also known as succinyl thiokinase) reaction, the hydrolysis of the energy-rich thioester bond in succinyl-CoA is coupled to the synthesis of GTP. This is yet another example of substrate-level phosphorylation, in which a high-energy bond in a metabolic intermediate is used for the synthesis of an energy-rich nucleotide. GTP is equivalent to ATP, with which it is in equilibrium through the nucleoside diphosphate kinase reaction:

Succinate is a four-carbon dicarboxylic acid. In the remaining reactions, two hydrogen atoms of succinate are replaced by oxygen to complete the cycle with the formation of oxaloacetate.

The enzyme succinate dehydrogenase (SDH) forms fumarate by transferring two hydrogen atoms from succinate to its prosthetic group FAD and from FADH₂

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