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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 16.1Oxidation of Glucose and Fatty Acids to CO2

The complete aerobic oxidation of glucose is coupled to the synthesis of as many as 36 molecules of ATP:

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Glycolysis, the initial stage of glucose metabolism, takes place in the cytosol and does not involve molecular O2. It produces a small amount of ATP and the three-carbon compound pyruvate. In aerobic cells, pyruvate formed in glycolysis is transported into the mitochondria, where it is oxidized by O2 to CO2. Via chemiosmotic coupling, the oxidation of pyruvate in the mitochondria generates the bulk of the ATP produced during the conversion of glucose to CO2. In this section, we discuss the biochemical pathways that oxidize glucose and fatty acids to CO2 and H2O; the fate of the released electrons is described in the next section.

Cytosolic Enzymes Convert Glucose to Pyruvate

A set of 10 enzymes catalyze the reactions, constituting the glycolytic pathway, that degrade one molecule of glucose to two molecules of pyruvate (Figure 16-3). All the metabolic intermediates between glucose and pyruvate are watersoluble phosphorylated compounds. Four molecules of ATP are formed from ADP in glycolysis (reactions 6 and 9). However, two ATP molecules are consumed during earlier steps of this pathway: the first by the addition of a phosphate residue to glucose in the reaction catalyzed by hexokinase (reaction 1), and the second by the addition of a second phosphate to fructose 6-phosphate in the reaction catalyzed by phosphofructokinase-1 (reaction 3). Thus there is a net gain of two ATP molecules.

Figure 16-3. The glycolytic pathway by which glucose is degraded to pyruvic acid.

Figure 16-3

The glycolytic pathway by which glucose is degraded to pyruvic acid. Reactions in which ATP and ADP are involved are highlighted in blue; the reaction involving NAD and NADH is highlighted in yellow. Note that all the intermediates between glucose and (more...)

The balanced chemical equation for the conversion of glucose to pyruvate shows that four hydrogen atoms (four protons and four electrons) are also formed:

Image ch16e3.jpg

(For convenience, we show pyruvate in its un-ionized form, pyruvic acid, although at physiological pH it would be largely dissociated.) All four electrons and two of the four protons are transferred to two molecules of the oxidized form of the electron carrier nicotinamide adenine dinucleotide (NAD+) to produce the reduced form, NADH (Figure 16-4):

Image ch16e4.jpg
The reaction that generates these hydrogen atoms and transfers them to NAD+ is catalyzed by glyceraldehyde 3-phosphate dehydrogenase (see Figure 16-3, reaction 5).

Figure 16-4. Structures of the electron-carrying coenzymes NAD+ and NADH.

Figure 16-4

Structures of the electron-carrying coenzymes NAD+ and NADH. Nicotinamide adenine dinucleotide (NAD+) and the related nicotinamide adenine dinucleotide phosphate (NADP+) accept only pairs of electrons; reduction to NADH or NADPH involves the transfer (more...)

Thus the overall reaction for this first stage of glucose metabolism is

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Substrate-Level Phosphorylation Generates ATP during Glycolysis

As noted earlier, the immediate energy source for ATP synthesis in chloroplasts and mitochondria is provided by the proton-motive force across a membrane. Cells also can produce ATP by a process called substrate-level phosphorylation, which is catalyzed by water-soluble enzymes in the cytosol; membranes and ion gradients are not involved.

Substrate-level phosphorylation occurs twice in the glycolytic pathway. The first results from the pair of reactions that convert glyceraldehyde 3-phosphate to 3-phosphoglycerate (see Figure 16-3, steps 5 and 6). In the first of these reactions, oxidation of the aldehyde (CHO) group on glyceraldehyde 3-phosphate by NAD+ is coupled to addition of a phosphate group, forming 1,3-bisphosphoglycerate with a single high-energy phosphoanhydride bond to carbon 1. In this reaction, catalyzed by glyceraldehyde 3-phosphate dehydrogenase, a high-energy thioester enzyme intermediate is formed (Figure 16-5). The high-energy phosphate group of 1,3-bisphosphoglycerate then is transferred to ADP, forming ATP and 3-phosphoglycerate, a reaction catalyzed by phosphoglycerate kinase. Although the first reaction requires free energy (ΔG°′ = +1.5 kcal/mol), the second releases even more free energy (ΔG°′ = −4.5 kcal/mol). Thus the net change in standard free energy of these two reactions is −3.0 kcal/mol, so the two reactions overall are strongly exergonic.

Figure 16-5. The mechanism of action of glyceraldehyde 3-phosphate dehydrogenase.

Figure 16-5

The mechanism of action of glyceraldehyde 3-phosphate dehydrogenase. The sulfhydryl (SH) group is the side chain of cysteine at the active site of the enzyme; R symbolizes the rest of the glyceraldehyde 3-phosphate molecule. R′ is the rest of (more...)

The three final reactions of glycolysis, in which 3-phosphoglycerate is converted to pyruvate, also result in substrate-level phosphorylation (see Figure 16-3, steps 7 – 9). The first two of these reactions, which each have a small positive ΔG°′, lead to formation of phosphoenolpyruvate. In the third reaction, catalyzed by pyruvate kinase, the high-energy phosphate group in phosphoenolpyruvate is transferred to ADP, yielding pyruvate and ATP. This reaction is strongly exergonic (ΔG°′ = −7.5 kcal/mol), as is the net free-energy change for the three reactions overall (ΔG°′ = −6.0 kcal/mol).

The glycolytic conversion of one molecule of glucose to two molecules of glyceraldehyde 3-phosphate consumes two ATPs (see Figure 16-3). The two subsequent substrate-level phosphorylations yield two ATPs for each molecule of glyceraldehyde, or a total of four ATPs per glucose molecule. Thus the net yield is two ATPs per glucose molecule. The remaining 34 molecules of ATP generated during the complete aerobic metabolism of glucose are synthesized during oxidation of pyruvate to CO2 and H2O in mitochondria. Before discussing what occurs in mitochondria, however, we digress briefly to describe the anaerobic metabolism of glucose.

Anaerobic Metabolism of Each Glucose Molecule Yields Only Two ATP Molecules

Most eukaryotes are obligate aerobes: they grow only in the presence of oxygen and metabolize glucose (or related sugars) completely to CO2, with the concomitant production of a large amount of ATP. Most eukaryotes, however, can generate some ATP by anaerobic metabolism. A few eukaryotes are facultative anaerobes: they grow in either the presence or the absence of oxygen. For example, annelids, mollusks, and some yeasts can live and grow for days without oxygen. Certain prokaryotes are obligate anaerobes: they cannot grow in the presence of oxygen, and they metabolize glucose only anaerobically.

In the absence of oxygen, glucose is not converted entirely to CO2 (as it is in obligate aerobes) but to one or more two- or three-carbon compounds, and only in some cases to CO2. For instance, yeasts degrade glucose to two pyruvate molecules via glycolysis, generating a net of two ATP and two NADH molecules per glucose molecule. If necessary, yeasts can anaerobically convert pyruvate to ethanol and CO2; two NADH molecules are oxidized to NAD+ for each two pyruvates converted to ethanol, thereby regenerating the supply of NAD+ (Figure 16-6, right). This anaerobic fermentation is the basis of beer and wine production.

Figure 16-6. The anaerobic metabolism of glucose.

Figure 16-6

The anaerobic metabolism of glucose. In the formation of pyruvate from glucose, one molecule of NAD+ is reduced to NADH for each molecule of pyruvate formed (see Figure 16-3). To regenerate NAD+, two electrons are transferred from each NADH molecule to (more...)

During the prolonged contraction of mammalian skeletal muscle cells, oxygen becomes limited and glucose cannot be oxidized completely to CO2 and H2O. In this situation, muscle cells ferment glucose to two molecules of lactic acid — again, with the net production of only two molecules of ATP per glucose molecule (Figure 16-6, left). The lactic acid causes muscle and joint aches. It is largely secreted into the blood; some passes into the liver, where it is reoxidized to pyruvate and either further metabolized to CO2 or converted to glucose. Much lactate is metabolized to CO2 by the heart, which is highly perfused by blood and can continue aerobic metabolism at times when exercising skeletal muscles secrete lactate.

Lactic acid bacteria (the organisms that “spoil” milk) and other prokaryotes also generate ATP by the fermentation of glucose to lactate.

Mitochondria Possess Two Structurally and Functionally Distinct Membranes

In the second stage of aerobic oxidation, pyruvate formed in glycolysis is transported into mitochondria, where it is oxidized by O2 to CO2. These mitochondrial oxidation reactions generate 34 of the 36 ATP molecules produced from the conversion of glucose to CO2. Mitochondria thus are the “power plants” of eukaryotic cells. To understand how mitochondria operate, we first need to be familiar with their structure.

Mitochondria are among the larger organelles in the cell, each one being about the size of an E. coli bacterium. Most eukaryotic cells contain many mitochondria, which collectively can occupy as much as 25 percent of the volume of the cytoplasm. They are large enough to be seen under a light microscope, but the details of their structure can be viewed only with the electron microscope (see Figure 5-45). The outer and the inner mitochondrial membranes define two submitochondrial compartments: the intermembrane space between the two membranes, and the matrix, or central compartment, (Figure 16-7). The fractionation and purification of these membranes and compartments has made it possible to determine their protein and phospholipid compositions and to localize each enzyme-catalyzed reaction to a specific membrane or space.

Figure 16-7. A three-dimensional diagram of a mitochondrion cut longitudinally.

Figure 16-7

A three-dimensional diagram of a mitochondrion cut longitudinally. The F0F1 complexes (small red spheres), which synthesize ATP, are intramembrane particles that protrude from the inner membrane into the matrix. The matrix contains the mitochondrial DNA (more...)

The outer membrane defines the smooth outer perimeter of the mitochondrion and contains mitochondrial porin, a transmembrane channel protein similar in structure to bacterial porins (see Figure 3-35). Ions and most small molecules (up to about 5000 MW) can readily pass through these channel proteins. Although the flow of metabolites across the outer membrane may limit their rate of mitochondrial oxidation, the inner membrane is the major permeability barrier between the cytosol and the mitochondrial matrix.

Freeze-fracture studies indicate that the inner membrane contains many protein-rich intramembrane particles. Some are the F0F1 complexes that synthesize ATP; others function in transporting electrons to O2 from NADH or reduced flavin adenine dinucleotide (FADH2) (Figure 16-8). Various transport proteins located in the inner membrane allow otherwise impermeable molecules, such as ADP and Pi, to pass from the cytosol to the matrix, and other molecules, such as ATP, to move from the matrix into the cytosol. Protein constitutes 76 percent of the total inner membrane weight — a higher fraction than in any other cellular membrane. Cardiolipin (diphosphatidylglycerol), a lipid concentrated in the inner membrane, sufficiently reduces the membrane’s permeability to protons that a proton-motive force can be established across it.

Figure 16-8. Structure of FAD and its reduction to FADH2.

Figure 16-8

Structure of FAD and its reduction to FADH2. The coenzyme flavin adenine dinucleotide (FAD) can accept one or two hydrogen atoms. The addition of one electron together with a proton (i.e., a hydrogen atom) generates a semiquinone intermediate. The semiquinone (more...)

The mitochondrial inner membrane and matrix are the sites of most reactions involving the oxidation of pyruvate and fatty acids to CO2 and H2O and the coupled synthesis of ATP from ADP and Pi. These complex processes involve many steps but can be subdivided into three groups of reactions, each of which occurs in a discrete membrane or space in the mitochondrion (Figure 16-9):

Figure 16-9. Summary of the aerobic oxidation of pyruvate in mitochondria.

Figure 16-9

Summary of the aerobic oxidation of pyruvate in mitochondria. The outer membrane is not shown because it is freely permeable to all metabolites. Specific transport proteins (ovals) in the inner membrane import pyruvate (tan), ADP (green), and Pi (purple) (more...)

1.

Oxidation of pyruvate and fatty acids to CO2, coupled to the reduction of the coenzymes NAD+ and FAD to NADH and FADH2, respectively. These reactions occur in the matrix or on inner-membrane proteins facing it.

2.

Electron transfer from NADH and FADH2 to O2. These reactions occur in the inner membrane and are coupled to the generation of a proton-motive force across it.

3.

Harnessing of the energy stored in the electrochemical proton gradient for ATP synthesis by the F0F1 complex in the inner membrane.

The inner mitochondrial membrane has numerous infoldings, or cristae, that greatly expand its surface area, enhancing its ability to generate ATP (see Figure 16-7). In typical liver mitochondria, for example, the area of the inner membrane is about five times that of the outer membrane. In fact, the total area of all inner mitochondrial membranes in liver cells is about 17 times that of the plasma membrane. The mitochondria in heart and skeletal muscles contain three times as many cristae as are found in typical liver mitochondria— presumably reflecting the greater demand for ATP by muscle cells.

Image plant.jpgIn plants, stored carbohydrates, mostly in the form of starch, are hydrolyzed to glucose. Glycolysis then produces pyruvate, which is transported into mitochondria, as in animal cells. Mitochondrial oxidation of pyruvate and concomitant formation of ATP occurs in photosynthetic cells during dark periods when photosynthesis is not possible, and in roots and other nonphotosynthetic tissues all the time.

Mitochondrial Oxidation of Pyruvate Begins with the Formation of Acetyl CoA

Immediately after pyruvate is transported from the cytosol across the mitochondrial membranes to the matrix, it reacts with coenzyme A, forming CO2 and the intermediate acetyl CoA (Figure 16-10). This reaction, catalyzed by pyruvate dehydrogenase, is highly exergonic (ΔG°′ = −8.0 kcal/mol) and essentially irreversible. Pyruvate dehydrogenase, which is located in the mitochondrial matrix, is a giant multienzyme complex 30 nm in diameter (4.6 × 106 MW), even larger than a ribosome. Composed of three different enzymes, pyruvate dehydrogenase contains 60 subunits, several regulatory polypeptides, and five different coenzymes (Figure 16-11).

Figure 16-10. The structure of acetyl CoA.

Figure 16-10

The structure of acetyl CoA. This compound is an important intermediate in the aerobic oxidation of pyruvate, fatty acids, and many amino acids. It also contributes acetyl groups in many biosynthetic pathways.

Figure 16-11. Structure of pyruvate dehydrogenase and its catalytic activities.

Figure 16-11

Structure of pyruvate dehydrogenase and its catalytic activities. (a) This very large complex contains three distinct enzymes: E1 is pyruvate decarboxylase (24 subunits); E2 is lipoamide transacetylase (24 subunits); and E3 is dihydrolipoyl dehydrogenase (more...)

As discussed later, acetyl CoA plays a central role in the oxidation of fatty acids and many amino acids. In addition, it is an intermediate in numerous biosynthetic reactions, such as the transfer of an acetyl group to lysine residues in histone proteins and to the N-termini of many mammalian proteins. Acetyl CoA also is a biosynthetic precursor of cholesterol and other steroids, and of the farnesyl and related groups that anchor proteins such as Ras to membranes (see Figure 3-36b). In respiring mitochondria, however, the acetyl group of acetyl CoA is almost always oxidized to CO2.

Oxidation of the Acetyl Group of Acetyl CoA in the Citric Acid Cycle Yields CO2 and Reduced Coenzymes

The final stage in the oxidation of glucose entails a set of nine reactions in which the acetyl group of acetyl CoA is oxidized to CO2. These reactions operate in a cycle that is referred to by several names: the citric acid cycle, the tricarboxylic acid cycle, and the Krebs cycle. The net result is that for each acetyl group entering the cycle as acetyl CoA, two molecules of CO2 are produced:

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As shown in Figure 16-12, the cycle begins with the condensation of the two-carbon acetyl group from acetyl CoA with the four-carbon molecule oxaloacetate to yield the six-carbon citric acid. In both reactions 4 and 5, a CO2 molecule is released; in reactions 4, 5, 7, and 9, oxidation of cycle intermediates generates reduced electron carriers (three NADH molecules and one FADH2 molecule). In reaction 6, hydrolysis of the high-energy thioester bond in succinyl CoA is coupled to synthesis of one GTP by substrate-level phosphorylation. (GTP and ATP are interconvertible). The final reaction (9) also regenerates oxaloacetate, so the cycle can begin again. Note that molecular O2 does not participate in the citric acid cycle.

Figure 16-12. The citric acid cycle, in which acetyl groups transferred from acetyl CoA are oxidized to CO2.

Figure 16-12

The citric acid cycle, in which acetyl groups transferred from acetyl CoA are oxidized to CO2. In reaction 1, a two-carbon acetyl residue from acetyl CoA condenses with the four-carbon molecule oxaloacetate to form the six-carbon molecule citrate. In (more...)

Most enzymes and small molecules involved in the citric acid cycle are soluble in aqueous solution and are localized to the matrix of the mitochondrion. This includes the water-soluble molecules CoA, acetyl CoA, and succinyl CoA, as well as NAD+ and NADH. Succinate dehydrogenase together with FAD/FADH2 (reaction 7) and α-ketoglutarate dehydrogenase (reaction 5) are localized to the inner membrane with active sites facing the matrix.

The protein concentration of the mitochondrial matrix is 500 mg/ml (a 50 percent protein solution), and the matrix must have a viscous, gel-like consistency. When mitochondria are disrupted by gentle ultrasonic vibration or osmotic lysis, the six non-membrane-bound enzymes in the citric acid cycle are released as a very large multiprotein complex. The reaction product of one enzyme, it is believed, passes directly to the next enzyme without diffusing through the solution. However, much work is needed to determine the structure of the enzyme complex: biochemists generally study the properties of enzymes in dilute aqueous solutions of less than 1 mg/ml, and weak interactions between enzymes are often difficult to detect.

Since glycolysis of one glucose molecule generates two acetyl CoA molecules, the reactions in the glycolytic pathway and citric acid cycle produce six CO2 molecules, 10 NADH molecules, and two FADH2 molecules per glucose molecule (Table 16-1). Although these reactions also generate four high-energy phosphoanhydride bonds in the form of two ATP and two GTP molecules, this represents only a small fraction of the available energy released in the complete aerobic oxidation of glucose. The remaining energy is stored in the reduced coenzymes, NADH and FADH2. Synthesis of most of the ATP generated in aerobic oxidation is coupled to the reoxidation of these compounds by O2 in a stepwise process involving the electron transport chain. Moreover, even though molecular O2 is not involved in any reaction of the citric acid cycle, in the absence of O2 the cycle soon stops operating as the supply of NAD+ and FAD dwindles. Before considering electron transport and the coupled formation of ATP in detail, we first discuss how the supply of NAD+ in the cytosol is regenerated and then the oxidation of fatty acids to CO2.

Table 16-1. Net Result of the Glycolytic Pathway and the Citric Acid Cycle.

Table 16-1

Net Result of the Glycolytic Pathway and the Citric Acid Cycle.

Inner-Membrane Proteins Allow the Uptake of Electrons from Cytosolic NADH

For aerobic oxidation to continue, the NADH produced during glycolysis in the cytosol must be regenerated. As with NADH generated in the mitochondrial matrix, electrons from cytosolic NADH are ultimately transferred to O2 via the electron transport chain, concomitant with the generation of a proton-motive force. Although the inner mitochondrial membrane is impermeable to NADH itself, several electron shuttles can transfer electrons from cytosolic NADH to the matrix.

In the most widespread shuttle — the malate shuttle —  cytosolic NADH reduces oxaloacetate to malate (Figure 16-13, reaction 1). An antiport protein in the mitochondrial inner membrane then transports malate into the matrix in exchange for α-ketoglutarate (reaction 2). A soluble dehydrogenase in the matrix then converts malate to oxaloacetate, reducing NAD+ to NADH in the process (reaction 3). Since oxaloacetate, an intermediate in the Krebs cycle cannot cross the inner membrane, it is first converted to the amino acid aspartate, which crosses the inner membrane in exchange for glutamate (reactions 4 and 5). Once in the cytosol, the aspartate is reconverted to oxaloacetate in a reaction in which α-ketoglutarate is converted to glutamate (reaction 6). The net effect of this complex cycle is the oxidation of cytosolic NADH to NAD+, together with the reduction of matrix NAD+ to NADH.

Figure 16-13. The malate shuttle.

Figure 16-13

The malate shuttle. Because the inner mitochondrial membrane is impermeable to NADH, the cell uses an indirect mechanism to transfer electrons from cytosolic NADH to NAD+ in the matrix. Two antiport proteins in the membrane and two soluble enzymes present (more...)

Mitochondrial Oxidation of Fatty Acids Is Coupled to ATP Formation

Fatty acids are stored as triacylglycerols, primarily as droplets in adipose (fat-storing) cells. In response to hormones such as adrenaline, triacylglycerols are hydrolyzed in the cytosol to free fatty acids and glycerol:

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Fatty acids are released into the blood, from which they are taken up and oxidized by most cells. They are the major energy source for many tissues, particularly heart muscle. In humans, the oxidation of fats is quantitatively more important than the oxidation of glucose as a source of ATP. In part, this is because the oxidation of 1 g of triacylglycerol to CO2 generates about six times as much ATP as does the oxidation of 1 g of hydrated glycogen, the polymeric storage form of glucose in animals.

In the cytosol, free fatty acids are linked to coenzyme A to form a fatty acyl CoA in an exergonic reaction coupled to the hydrolysis of ATP to AMP and PPi (inorganic pyrophosphate):

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Subsequent hydrolysis of PPi to two molecules of phosphate (Pi) drives this reaction to completion. Then the fatty acyl group is transferred to carnitine, moved across the inner mitochondrial membrane by a transporter protein, and is released from carnitine and reattached to another CoA molecule on the matrix side. Each molecule of a fatty acyl CoA in the mitochondrion is oxidized to form one molecule of acetyl CoA and an acyl CoA shortened by two carbon atoms (Figure 16-14). Concomitantly, one molecule apiece of NAD+ and FAD are reduced, respectively, to NADH and FADH2. This set of reactions is repeated on the shortened acyl CoA until all the carbon atoms are converted to acetyl CoA.

Figure 16-14. Oxidation of fatty acids in mitochondria.

Figure 16-14

Oxidation of fatty acids in mitochondria. Four enzyme-catalyzed reactions convert a fatty acyl CoA molecule to acetyl CoA and a fatty acyl CoA shortened by two carbon atoms. Concomitantly, one NAD+ molecule is reduced to NADH and one FAD molecule is reduced (more...)

For example, mitochondrial oxidation of each molecule of the 18-carbon stearic acid, CH3(CH2)16COOH, yields nine molecules of acetyl CoA and eight molecules each of NADH and FADH2. As with acetyl CoA generated from pyruvate, these acetyl groups enter the citric acid cycle and are oxidized to CO2. Electrons from the reduced coenzymes produced in the oxidation of fatty acyl CoA to acetyl CoA and in the subsequent oxidation of acetyl CoA in the citric acid cycle move via the electron transport chain to O2, coupled to regeneration of a proton-motive force that is used to power ATP synthesis (see Figure 16-9).

Oxidation of Fatty Acids in Peroxisomes Generates No ATP

Mitochondrial oxidation of fatty acids is the major source of ATP in mammalian liver cells, and biochemists at one time believed this was true in all cell types. However, rats treated with clofibrate, a drug used to reduce the level of blood lipoproteins, were found to exhibit an increased rate of fatty acid oxidation and a large increase in the number of peroxisomes in their liver cells. This finding suggested that peroxisomes, as well as mitochondria, can oxidize fatty acids. These small organelles, ≈0.2 – 1 μm in diameter, are lined by a single membrane (see Figure 5-47). Also called microbodies, peroxisomes are present in all mammalian cells except erythrocytes and are also found in plant cells, yeasts, and probably most eukaryotic cells. The peroxisome is now recognized as the principal organelle in which fatty acids are oxidized in most cell types. Indeed, very long chain fatty acids containing more than about 20 CH2 groups are degraded only in peroxisomes; in mammalian cells, mid-length fatty acids containing 10 – 20 CH2 groups can be degraded in both peroxisomes and mitochondria.

Peroxisomes contain several oxidases — enzymes that use oxygen as an electron acceptor to oxidize organic substances, in the process forming hydrogen peroxide (H2O2), which is then degraded by catalase:

Image ch16e9.jpg

The pathway of peroxisomal degradation of fatty acids is similar to that used in liver mitochondria. However, peroxisomes lack an electron transport chain, and electrons from the FADH2 and NADH produced during the oxidation of fatty acids are immediately transferred to O2, regenerating FAD and NAD+, and forming H2O2 (Figure 16-15). Catalase quickly decomposes the H2O2, which is highly toxic to the cell.

Figure 16-15. Oxidation of fatty acids by peroxisomes.

Figure 16-15

Oxidation of fatty acids by peroxisomes. Peroxisomes degrade fatty acids with more than 12 carbon atoms by a series of reactions similar to those used by liver mitochondria (see Figure 16-14). In peroxisomes, however, the electrons and protons transferred (more...)

In contrast to mitochondrial fatty acid oxidation, which is coupled to generation of ATP, peroxisomal oxidation of fatty acids is not linked to ATP formation, and the released energy is converted to heat. The acetyl group of acetyl CoA generated during peroxisomal oxidation of fatty acids (see Figure 16-15) is transported into the cytosol, where it is used in the synthesis of cholesterol and other metabolites.

Image med.jpgBefore fatty acids can be degraded in the peroxisome, they must first be transported into the organelle from the cytosol. Mid-length fatty acids are esterified to coenzyme A in the cytosol; the resulting fatty acyl CoA is then transported into the peroxisome by a specific transporter. However, very long chain fatty acids enter the peroxisome by another transporter, and then are esterified to CoA once inside. In the human genetic disease X-linked adrenoleukodystrophy (ALD), peroxisomal oxidation of very long chain fatty acids is specifically defective, while the oxidation of mid-length fatty acids is normal. In ALD, very long chain fatty acids are transported normally into peroxisomes, but are not esterified to CoA and so cannot be oxidized. The enzyme that catalyzes this esterification is synthesized in the cytosol; as we discuss in Chapter 17, the ADL gene encodes the peroxisomal membrane protein required for uptake of this enzyme into peroxisomes. Patients with the severe form of ADL are unaffected until mid-childhood, when severe neurological disorders appear, followed by death within a few years.

The Rate of Glucose Oxidation Is Adjusted to Meet the Cell’s Need for ATP

All enzyme-catalyzed reactions and metabolic pathways are regulated by cells so as to produce the needed amounts of metabolites but not an excess. The primary function of the oxidation of glucose to CO2 in the glycolytic pathway and the citric acid cycle is to produce NADH and FADH2, whose oxidation in the mitochondria generates ATP. The opera-tion of both pathways is continuously regulated, primarily by allosteric mechanisms, to meet the cell’s need for ATP (Chapter 3).

Three glycolytic enzymes that are allosterically controlled play a key role in regulating the entire glycolytic pathway (see Figure 16-3). Hexokinase, which catalyzes the first step, is inhibited by its reaction product, glucose 6-phosphate. Pyruvate kinase, which catalyzes the last step, is inhibited by ATP, so glycolysis slows down if too much ATP is present. The third enzyme, phosphofructokinase-1, catalyzes the third reaction in the conversion of glucose to pyruvate and is the principal rate-limiting enzyme of the glycolytic pathway. Emblematic of its critical role in regulating the rate of glycolysis, this enzyme is controlled by four allosteric molecules (Figure 16-16).

Figure 16-16. Enzymatic control of glucose metabolism in the cytosol.

Figure 16-16

Enzymatic control of glucose metabolism in the cytosol. Phosphofructokinase-1 is the main control point in the regulation of the glycolytic pathway. It is allosterically inhibited by ATP and citrate, and stimulated by ADP and fructose 2,6-bisphosphate. (more...)

If citrate — the product of the first step of the citric acid cycle — accumulates, it allosterically inhibits the activity of phosphofructokinase-1, thereby reducing the generation of pyruvate and acetyl CoA, so that less citrate is formed. This feedback inhibition of phosphofructokinase-1 by citrate allows the activities of the glycolytic pathway to be coordinated with those of the citric acid cycle. Intermediates in the citric acid cycle are also used in biosynthesis of amino acids; a buildup of citrate indicates that these intermediates are plentiful and that glucose need not be degraded for this purpose.

Phosphofructokinase-1 also is allosterically activated by ADP and allosterically inhibited by ATP. This arrangement makes the rate of glycolysis very sensitive to intracellular levels of ATP and ADP. The allosteric inhibition of phosphofructokinase-1 by ATP may seem unusual, since ATP is also a substrate of this enzyme. But the affinity of the substrate-binding site for ATP is much higher (has a lower Km) than that of the allosteric site. Thus at low concentrations, ATP binds to the catalytic but not to the inhibitory allosteric site and enzymatic catalysis proceeds at near maximal rates. At high concentrations, ATP binds to the allosteric site, inducing a conformational change that reduces the affinity of the enzyme for the other substrate, fructose 6-phosphate, and thus inhibits the rate of this reaction and the overall rate of glycolysis.

The metabolite fructose 2,6-bisphosphate is another important allosteric activator of phosphofructokinase-1 (see Figure 16-16). Fructose 2,6-bisphosphate is formed from the glycolytic intermediate fructose 6-phosphate; the catalyst is phosphofructokinase-2, an enzyme different from phosphofructokinase-1. Fructose 6-phosphate accelerates the formation of fructose 2,6-bisphosphate, which, in turn, activates phosphofructokinase-1. This type of control, by analogy with feedback control, is known as feed-forward activation, in which the abundance of a metabolite (here, fructose 6-phosphate) induces an acceleration in its metabolism. Fructose 2,6-bisphosphate allosterically activates phosphofructokinase-1 in liver cells by decreasing the inhibitory effect of ATP and by increasing the affinity of phosphofructokinase-1 for one of its substrates, fructose 6-phosphate.

The three glycolytic enzymes that are regulated by allosteric molecules catalyze reactions with large negative ΔG°′ values — reactions that are essentially irreversible under ordinary conditions. These enzymes thus are particularly suitable for regulating the entire glycolytic pathway. Additional control is exerted by glyceraldehyde 3-phosphate dehydrogenase, which catalyzes the reduction of NAD+ to NADH (see Figure 16-3). If cytosolic NADH builds up owing to a slowdown in mitochondrial oxidation, this step in glycolysis will be slowed by mass action. As we discuss later, mitochondrial oxidation of NADH and FADH2, produced in the glycolytic pathway and citric acid cycle, also is tightly controlled to produce the appropriate amount of ATP required by the cell.

Glucose metabolism is controlled differently in various mammalian tissues to meet the metabolic needs of the organism as a whole. During periods of carbohydrate starvation, for instance, glycogen in the liver is converted directly to glucose 6-phosphate (without involvement of lexokinase). Under these conditions, however, phosphofructokinase-1 is inhibited and thus glucose 6-phosphate is not metabolized to pyruvate; rather, it is converted to glucose by a phosphatase and released into the blood to nourish the brain and muscles, which then oxidize the bulk of the available glucose. (Chapter 20 contains a more detailed discussion of the control of glucose metabolism in the liver and muscles.) In all cases, the activity of these enzymes is regulated by the level of small-molecule metabolites, generally by allosteric interactions or by phosphorylation.

Additional control of glucose oxidation occurs in the mitochondria. Pyruvate dehydrogenase is deactivated by phosphorylation, which is stimulated by high levels of ATP, NADH, and acetyl CoA. Three enzymes of the citric acid cycle also are regulated. As a consequence of these multiple sites of regulation, the entry of two-carbon units into the citric acid cycle and the rate of the cycle are decreased when the cell has sufficient ATP. In this case, extra acetyl CoA is used to synthesize fatty acids, which are stored as triacylglycerols in adipose tissue.

SUMMARY

  •  In the cytosol of eukaryotic cells, glucose is converted to pyruvate via the glycolytic pathway, with the net formation of two ATPs and the net reduction of two NAD+ molecules to NADH (see Figure 16-3). ATP is formed by two substrate-level phosphorylation reactions in the conversion of glyceraldehyde 3-phosphate to pyruvate.
  •  In anaerobic cells, pyruvate can be metabolized further to lactate or to ethanol plus CO2, with the reoxidation of NADH.
  •  Mitochondria have a permeable outer membrane and an inner membrane, which is the site of electron transport and ATP synthesis (see Figure 16-9).
  •  Pyruvate dehydrogenase, a very large, multienzyme complex in the mitochondrial matrix converts pyruvate into acetyl CoA and CO2 (see Figure 16-11).
  •  In each turn of the citric acid cycle, acetyl CoA condenses with the four-carbon molecule oxaloacetate to form the six-carbon citrate, which is converted back to oxaloacetate by a series of reactions that release two molecules of CO2 and generate three NADH molecules and one FADH2 molecule (see Figure 16-12).
  •  The NADH generated in the cytosol during glycolysis is oxidized to NAD+, with the concomitant reduction of NAD+ to NADH in the mitochondrial matrix, by a set of enzymes and transport proteins that form an electron shuttle.
  •  Electrons from NADH and FADH2 move via a series of membrane-bound electron carriers in the inner mitochondrial membrane to O2, regenerating NAD+ and FAD. This stepwise movement of electrons is coupled to pumping of protons across the inner membrane. The resulting proton-motive force powers ATP synthesis and generates most of the ATP resulting from aerobic oxidation of glucose.
  •  Oxidation of fatty acids in mitochondria yields acetyl CoA, which enters the citric acid cycle, and the reduced coenzymes NADH and FADH2. Subsequent oxidation of acetyl CoA and the reduced coenzymes is coupled to the formation of a proton-motive force that powers ATP formation.
  •  In most eukaryotic cells, oxidation of fatty acids, especially very long chain fatty acids, occurs primarily in peroxisomes and is not linked to ATP production; the released energy is converted to heat. The electrons released during peroxisomal oxidation of fatty acids are used to form H2O2, which is decomposed to H2O and O2 by catalase.
  •  The rate of glycolysis and the citric acid cycle, which depends on the cell’s need for ATP, is controlled by the inhibition and stimulation of several enzymes (see Figure 16-16). This complex regulation coordinates the activities of the glycolytic pathway and the citric acid cycle and results in the storage of glucose (as glycogen) or fat when ATP is abundant.
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Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21624