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

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Biochemistry. 5th edition.

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Section 16.3Glucose Can Be Synthesized from Noncarbohydrate Precursors

We now turn to the synthesis of glucose from noncarbohydrate precursors, a process called gluconeogenesis. This metabolic pathway is important because the brain depends on glucose as its primary fuel and red blood cells use only glucose as a fuel. The daily glucose requirement of the brain in a typical adult human being is about 120 g, which accounts for most of the 160 g of glucose needed daily by the whole body. The amount of glucose present in body fluids is about 20 g, and that readily available from glycogen, a storage form of glucose (Section 21.1), is approximately 190 g. Thus, the direct glucose reserves are sufficient to meet glucose needs for about a day. During a longer period of starvation, glucose must be formed from noncarbohydrate sources (Section 30.3.1).

The gluconeogenic pathway converts pyruvate into glucose. Noncarbohydrate precursors of glucose are first converted into pyruvate or enter the pathway at later intermediates such as oxaloacetate and dihydroxyacetone phosphate (Figure 16.24). The major noncarbohydrate precursors are lactate, amino acids, and glycerol. Lactate is formed by active skeletal muscle when the rate of glycolysis exceeds the rate of oxidative metabolism. Lactate is readily converted into pyruvate by the action of lactate dehydrogenase (Section 16.1.9). Amino acids are derived from proteins in the diet and, during starvation, from the breakdown of proteins in skeletal muscle (Section 30.3.1). The hydrolysis of triacylglycerols (Section 22.2.1) in fat cells yields glycerol and fatty acids. Glycerol is a precursor of glucose, but animals cannot convert fatty acids into glucose, for reasons that will be discussed later (Section 22.3.7). Glycerol may enter either the gluconeogenic or the glycolytic pathway at dihydroxyacetone phosphate.

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Figure 16.24. Pathway of Gluconeogenesis.

Figure 16.24

Pathway of Gluconeogenesis. The distinctive reactions and enzymes of this pathway are shown in red. The other reactions are common to glycolysis. The enzymes for gluconeogenesis are located in the cytosol, except for pyruvate carboxylase (in the mitochondria) (more...)

The major site of gluconeogenesis is the liver, with a small amount also taking place in the kidney. Little gluconeogenesis takes place in the brain, skeletal muscle, or heart muscle. Rather, gluconeogenesis in the liver and kidney helps to maintain the glucose level in the blood so that brain and muscle can extract sufficient glucose from it to meet their metabolic demands.

16.3.1. Gluconeogenesis Is Not a Reversal of Glycolysis

In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is converted into glucose. However, gluconeogenesis is not a reversal of glycolysis. Several reactions must differ because the equilibrium of glycolysis lies far on the side of pyruvate formation. The actual ΔG for the formation of pyruvate from glucose is about -20 kcal mol-1 (-84 kJ mol-1) under typical cellular conditions. Most of the decrease in free energy in glycolysis takes place in the three essentially irreversible steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.

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In gluconeogenesis, the following new steps bypass these virtually irreversible reactions of glycolysis:


Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.

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Fructose 6-phosphate is formed from fructose 1,6-bisphosphate by hydrolysis of the phosphate ester at carbon 1. Fructose 1,6-bisphosphatase catalyzes this exergonic hydrolysis.

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Glucose is formed by hydrolysis of glucose 6-phosphate in a reaction catalyzed by glucose 6-phosphatase.

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We will examine each of these steps in turn.

16.3.2. The Conversion of Pyruvate into Phosphoenolpyruvate Begins with the Formation of Oxaloacetate

The first step in gluconeogenesis is the carboxylation of pyruvate to form oxaloacetate at the expense of a molecule of ATP. Then, oxaloacetate is decarboxylated and phosphorylated to yield phosphoenolpyruvate, at the expense of the high phosphoryl-transfer potential of GTP. Both of these reactions take place inside the mitochondria.

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The first reaction is catalyzed by pyruvate carboxylase and the second by phosphoenolpyruvate carboxykinase. The sum of these reactions is:

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Pyruvate carboxylase is of special interest because of its structural, catalytic, and allosteric properties. The N-terminal 300 to 350 amino acids form an ATP-grasp domain (Figure 16.25), which is a widely used ATP-activating domain to be discussed in more detail when we investigate nucleotide biosynthesis (Section 25.1.1). The C-terminal 80 amino acids constitute a biotin-binding domain (Figure 16.26) that we will see again in fatty acid synthesis (Section 22.4.1). Biotin is a covalently attached prosthetic group, which serves as a carrier of activated CO2. The carboxylate group of biotin is linked to the ε-amino group of a specific lysine residue by an amide bond (Figure 16.27). Note that biotin is attached to pyruvate carboxylase by a long, flexible chain.

Figure 16.25. Domain Structure of Pyruvate Carboxylase.

Figure 16.25

Domain Structure of Pyruvate Carboxylase. The ATP-grasp domain activates HCO3- and transfers CO2 to the biotin-binding domain. From there, the CO2 is transferred to pyruvate generated in the central domain.

Figure 16.26. Biotin-Binding Domain of Pyruvate Carboxylase.

Figure 16.26

Biotin-Binding Domain of Pyruvate Carboxylase. Image mouse.jpg This likely structure is based on the structure of the homologous domain from the enzyme acetyl CoA carboxylase (Section 22.4.1). The biotin is on a flexible tether, allowing it to move between the ATP-bicarbonate (more...)

Figure 16.27. Structure of Carboxybiotin.

Figure 16.27

Structure of Carboxybiotin.

The carboxylation of pyruvate takes place in three stages:

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Recall that, in aqueous solutions, CO2 exists as HCO3- with the aid of carbonic anhydrase (Section 9.2). The HCO3- is activated to carboxyphosphate. This activated CO2 is subsequently bonded to the N-1 atom of the biotin ring to form the carboxybiotin-enzyme intermediate (see Figure 16.27). The CO2 attached to the biotin is quite activated. The ΔG°´ for its cleavage

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is -4.7 kcal mol-1 (-20 kJ mol-1). This negative ΔG°´ indicates that carboxybiotin is able to transfer CO2 to acceptors without the input of additional free energy.

The activated carboxyl group is then transferred from carboxybiotin to pyruvate to form oxaloacetate. The long, flexible link between biotin and the enzyme enables this prosthetic group to rotate from one active site of the enzyme (the ATP-bicarbonate site) to the other (the pyruvate site).

The first partial reaction of pyruvate carboxylase, the formation of carboxybiotin, depends on the presence of acetyl CoA. Biotin is not carboxylated unless acetyl CoA is bound to the enzyme. Acetyl CoA has no effect on the second partial reaction. The allosteric activation of pyruvate carboxylase by acetyl CoA is an important physiological control mechanism that will be discussed in Section 17.3.1.

16.3.3. Oxaloacetate Is Shuttled into the Cytosol and Converted into Phosphoenolpyruvate

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are cytoplasmic. Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol (Figure 16.28).

Figure 16.28. Compartmental Cooperation.

Figure 16.28

Compartmental Cooperation. Oxaloacetate utilized in the cytosol for gluconeogenesis is formed in the mitochondrial matrix by carboxylation of pyruvate. Oxaloacetate leaves the mitochondrion by a specific transport system (not shown) in the form of malate, (more...)

Finally, oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase in the cytosol. The CO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. Recall that, in glycolysis, the presence of a phosphoryl group traps the unstable enol isomer of pyruvate as phosphoenolpyruvate (Section 16.1.7). In gluconeogenesis, the formation of the unstable enol is driven by decarboxylation—the oxidation of the carboxylic acid to CO2—and trapped by the addition of a phosphate to carbon 2 from GTP. The two-step pathway for the formation of phosphoenolpyruvate from pyruvate has a ΔG°´ of +0.2 kcal mol-1 (+0.13 kJ mol-1) in contrast with +7.5 kcal mol-1 (+31 kJ mol-1) for the reaction catalyzed by pyruvate kinase. The much more favorable ΔG°´ for the two-step pathway results from the use of a molecule of ATP to add a molecule of CO2 in the carboxylation step that can be removed to power the formation of phosphoenolpyruvate in the decarboxylation step. Decarboxylations often drive reactions otherwise highly endergonic. This metabolic motif is used in the citric acid cycle (Section 17.1), the pentose phosphate pathway (Section 20.3.1), and fatty acid synthesis (Section 22.4.3).

16.3.4. The Conversion of Fructose 1,6-bisphosphate into Fructose 6-phosphate and Orthophosphate Is an Irreversible Step

On formation, phosphoenolpyruvate is metabolized by the enzymes of glycolysis but in the reverse direction. These reactions are near equilibrium under intracellular conditions; so, when conditions favor gluconeogenesis, the reverse reactions will take place until the next irreversible step is reached. This step is the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi.

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The enzyme responsible for this step is fructose 1,6-bisphosphatase. Like its glycolytic counterpart, it is an allosteric enzyme that participates in the regulation of gluconeogenesis. We will return to its regulatory properties later in the chapter.

16.3.5. The Generation of Free Glucose Is an Important Control Point

The fructose 6-phosphate generated by fructose 1,6-bisphosphatase is readily converted into glucose 6-phosphate. In most tissues, gluconeogenesis ends here. Free glucose is not generated; rather, the glucose 6-phosphate is processed in some other fashion, notably to form glycogen. One advantage to ending gluconeogenesis at glucose 6-phosphate is that, unlike free glucose, the molecule cannot diffuse out of the cell. To keep glucose inside the cell, the generation of free glucose is controlled in two ways. First, the enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated. Second, the enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis—tissues that release glucose into the blood. These tissues are the liver and to a lesser extent the kidney.

This final step in the generation of glucose does not take place in the cytosol. Rather, glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed to glucose by glucose 6-phosphatase, which is bound to the membrane (Figure 16.29). An associated Ca2+-binding stabilizing protein is essential for phosphatase activity. Glucose and Pi are then shuttled back to the cytosol by a pair of transporters. The glucose transporter in the endoplasmic reticulum membrane is like those found in the plasma membrane (Section 16.2.4). It is striking that five proteins are needed to transform cytosolic glucose 6-phosphate into glucose.

Figure 16.29. Generation of Glucose from Glucose 6-Phosphate.

Figure 16.29

Generation of Glucose from Glucose 6-Phosphate. Several endoplasmic reticulum (ER) proteins play a role in the generation of glucose from glucose 6-phosphate. T1 transports glucose 6-phosphate into the lumen of the ER, whereas T2 and T3 transport Pi and (more...)

16.3.6. Six High Transfer Potential Phosphoryl Groups Are Spent in Synthesizing Glucose from Pyruvate

Conceptual Insights, Energetics of Glucose Metabolism

Image mouse.jpg See the section on gluconeogenesis in the Conceptual Insights module to review why and how gluconeogenesis must differ from the reversal of glycolysis.

The stoichiometry of gluconeogenesis is:

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In contrast, the stoichiometry for the reversal of glycolysis is:

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Note that six nucleotide triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are generated in glycolysis in the conversion of glucose into pyruvate. Thus, the extra cost of gluconeogenesis is four high phosphoryl-transfer potential molecules per molecule of glucose synthesized from pyruvate. The four additional high phosphoryl-transfer potential molecules are needed to turn an energetically unfavorable process (the reversal of glycolysis, ΔG°´ = + 20 kcal mol-1 [+84 kJ mol-1]) into a favorable one (gluconeogenesis, ΔG°´ = -9 kcal mol-1 [-38 kJ mol-1]). This is a clear example of the coupling of reactions: ATP hydrolysis is used to power an energetically unfavorable reaction.

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Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22591