Chapter 15 : The Citric Acid Cycle

The Energy of Oxidations in the Cycle Is Efficiently Conserved

We have now covered one complete turn of the citric acid cycle (Fig. 15-10). An acetyl group, containing two carbon atoms, was fed into the cycle by combining with oxaloacetate. Two carbon atoms emerged from the cycle as CO₂ with the oxidation of isocitrate and a-ketoglutarate, and at the end of the cycle a molecule of oxaloacetate was regenerated. Note that the two carbon atoms appearing as CO₂ are not the same two carbons that entered in the form of the acetyl group; additional turns around the cycle are required before the carbon atoms that entered as an acetyl group finally appear as CO₂ (Fig. 15-7).

Although the citric acid cycle itself directly generates only one molecule of ATP per turn (in the conversion of succinyl-CoA to succinate), the four oxidation steps in the cycle provide a large flow of electrons into the respiratory chain and thus eventually lead to formation of a large number of ATP molecules during oxidative phosphorylation.

Figure 15-10 Each turn of the citric acid cycle produces three NADH and one FADH₂, as well as one GTP (or ATP). Two CO₂ are released in oxidative decarboxylation reactions. (Here and in several following figures, all cycle reactions are shown in one direction only. Keep in mind that most of the reactions are actually reversible, as shown in Fig. 15-7. )

We saw in the previous chapter that the energy yield from glycolysis (in which one molecule of glucose is converted into two of pyruvate) is two ATP molecules from one of glucose. When both pyruvate molecules are completely oxidized to yield six CO₂ molecules in the reactions catalyzed by the pyruvate dehydrogenase complex and the enzymes of the citric acid cycle, and the electrons are transferred to O₂ via the respiratory chain, as many as 38 ATP are obtained per glucose (Table 15-2). In round numbers, this represents the conservation of 38 x 30.5 kJ/mol = 1,160 kJ/mol, or 40% of the theoretical maximum of 2,840 kJ/mol available from the complete oxidation of glucose. These calculations employ the standard free-energy changes; when corrected for the actual free energy required to form ATP within cells (Box 13-2; p. 375), the calculated efficiency of the process is even greater.

Table 15-2 The stoichiometry of coenzyme reduction and ATP formation in the aerobic oxidation of a molecule of glucose via glycolysis, the pyruvate dehydrogenase reaction, and the citric acid cycle
Reaction	Number of ATP or reduced coenzymes directly formed	Number of ATP ultimately formed*
Glucose glucose-6-phosphate	-1 ATP	-1
Fructose-6-phosphate fructose-1,6-bisphosphate	-1 ATP	-1
2 Glyceraldehyde-3-phosphate 2 1,3-bisphosphoglycerate	2 NADH	6
2 1,3-Bisphosphoglycerate 2 3-phosphoglycerate	2 ATP	2
Phosphoenolpyruvate 2 pyruvate	2 ATP	2
2 Pyruvate 2 acetyl-CoA	2 NADH	6
2 Isocitrate α a-ketoglutarate	2 NADH	6
2 α-Ketoglutarate 2 succinyl-CoA	2 NADH	6
2 Succinyl-CoA 2 succinate	2 ATP (or 2 GTP)	2
2 Succinate 2 fumarate	2 FADH2	4
2 Malate 2 oxaloacetate	2 NADH	6
Total		38

* This is calculated as 3 ATP per NADH and 2 ATP per FADH₂. A negative value indicates consumption.

How Was the Cyclic Nature of the Pathway Established?

The historical road that led to the understanding of this cyclic metabolic pathway is instructive; it illustrates the general strategies used by biochemists since early in this century to unravel complex metabolic relationships. The citric acid cycle was first postulated as the pathway of pyruvate oxidation in animal tissues by Hans Krebs, in 1937. The idea of the cycle came to him during a study of the effect of the anions of various organic acids on the rate of oxygen consumption during pyruvate oxidation by suspensions of minced pigeon-breast muscle. This muscle, used in flight, has a very high rate of respiration and was thus especially appropriate for the study of oxidative activity. Earlier investigators, particularly Albert Szent-Gyorgyi, had found that certain four-carbon dicarboxylic acids known to be present in animal tissuessuccinate, fumarate, malate, and oxaloacetate-stimulate the consumption of oxygen by muscle. Krebs confirmed this observation and found that they also stimulate the oxidation of pyruvate. Moreover, he found that oxidation of pyruvate by muscle is also stimulated by the six-carbon tricarboxylic acids citrate, cis-aconitate, and isocitrate, and the five-carbon a-ketoglutarate. No other naturally occurring organic acids that were tested possessed such activity. The stimulatory effect of the active acids was remarkable: the addition of even a small quantity of any one of them could promote the oxidation of many times that amount of pyruvate.

The second important observation made by Krebs was that malonate (p. 458), a close analog of succinate and a competitive inhibitor of succinate dehydrogenase, inhibits the aerobic utilization of pyruvate by muscle suspensions, regardless of which actiue organic acid is added. This indicated that succinate and succinate dehydrogenase must be essential components in the enzymatic reactions involved in the oxidation of pyruvate. Krebs further found that when malonate is used to inhibit the aerobic utilization of pyruvate by a suspension of muscle tissue, there is an accumulation of citrate, a-ketoglutarate, and succinate in the suspending medium. This suggested that citrate and a-ketoglutarate are normally precursors of succinate.

From these basic observations and other evidence, Krebs concluded that the active tricarboxylic and dicarboxylic acids listed above can be arranged in a logical chemical sequence. Because the incubation of pyruvate and oxaloacetate with ground muscle tissue resulted in accumulation of citrate in the medium, Krebs reasoned that this sequence functions in a circular rather than linear manner: its beginning and end are linked together. For the missing link that closes the circle he proposed the reaction

Pyruvate + oxaloacetate citrate + CO₂

(Notice that in this detail Krebs was originally incorrect.)

From these simple experiments and logical reasoning, Krebs postulated what he called the citric acid cycle as the main pathway for oxidation of carbohydrate in muscle. The citric acid cycle is also called the tricarboxylic acid cycle because for some years after Krebs postulated the cycle it was uncertain whether citrate or some other tricarboxylic acid such as isocitrate was the first product formed by reaction of pyruvate and oxaloacetate. In the years since its discovery, the citric acid cycle has been found to function not only in muscles but in virtually all tissues of aerobic animals and plants and in many aerobic microorganisms.

The citric acid cycle was first postulated from experiments carried out on suspensions of minced muscle tissue. Subsequently its details were worked out by study of the highly purified enzymes of the cycle. One might ask whether these enzymes really function in a cycle in intact living cells and whether the rate of the cycle is high enough to account for the overall rate of glucose oxidation in animal tissues. These questions have been studied by the use of isotopically labeled metabolites, such as pyruvate or acetate, in which the isotopes ¹³C or ¹⁴C are used to mark a given carbon atom in the molecule. Many stringent experiments with the isotope tracer technique have confirmed that the citric acid cycle does take place in living cells, and at a high rate.

Some of the earliest experiments with isotopes produced an unexpected result, however, which aroused considerable controversy about the pathway and mechanism of the citric acid cycle. In fact, these experiments at first seemed to show that citrate was not the first tricarboxylic acid to be formed. Box 15-2 gives some details of this episode in the scientific history of the cycle.

B O X 15-2

Is Citric Acid the First Tricarboxylic Acid Formed in the Cycle?

When the heavy-carbon isotope ¹³C and the radioactive carbon isotopes ¹¹C and ¹⁴C became available, they were very soon put to use to trace the pathway of carbon atoms through the citric acid cycle. In one such experiment, which initiated the controversy over the role of citric acid, acetate labeled in the carboxyl group (designated [1-¹⁴C]acetate) was incubated aerobically with an animal tissue preparation. Acetate is enzymatically converted into acetyl-CoA in animal tissues, and the pathway of the labeled carboxyl carbon of the acetyl group in the cycle reactions could be traced. α-Ketoglutarate was isolated from the tissue after incubation, then degraded by known chemical reactions to establish the position(s) of the isotopic carbon derived from carboxyl-labeled acetate. Condensation of unlabeled oxaloacetate with carboxyl-labeled acetate would be expected to produce citrate labeled in one of the two primary carboxyl groups (Fig. 1). Because citrate is a symmetric molecule, with no asymmetric carbon, its two terminal carboxyl groups are chemically indistinguishable. Therefore, half of the labeled citrate molecules were expected to yield a-ketoglutarate labeled in the α-carboxyl group and the other half to yield a-ketoglutarate labeled in the y-carboxyl group; that is, the α-ketoglutarate isolated should have been a mixture of molecules labeled in both carboxyl groups.

Contrary to this expectation, the labeled α-ketoglutarate isolated from the tissue suspension contained the isotope only in the γ-carboxyl group (Fig. 1). It was concluded that citrate itself, or any other symmetric molecule, could not possibly be an intermediate in the pathway from acetate to α-ketoglutarate. Hence an asymmetric tricarboxylic acid, presumably cis-aconitate or isocitrate, had to be the first condensation product formed from acetate and oxaloacetate.

In 1948, however, Alexander Ogston pointed out that although citrate has no chiral center (see Fig. 3-9), it has the potential of reacting asymmetrically if the enzyme acting upon it has an active site that is asymmetric. He suggested that the active site of aconitase, the enzyme acting on the newly formed citrate, may have three points to which the citrate molecule must be bound and that the citrate molecule must undergo a specific three-point attachment to these binding points. As seen in Figure 2, the binding of citrate to the three points can happen in only one way, and this would account for the formation of only one type of labeled α-ketoglutarate. Organic molecules such as citrate that have no chiral center, but are potentially capable of reacting asymmetrically with an asymmetric active site, are now called prochiral molecules.

Why Is the Oxidation of Acetate So Complicated?

This eight-step, cyclic process for oxidation of the simple two-carbon acetyl groups to CO₂ may seem unnecessarily cumbersome and not in keeping with the principle of maximum economy in the molecular logic of living cells. The role of the citric acid cycle is not confined to the oxidation of acetate, however; this pathway is the hub of intermediary metabolism. Four- and five-carbon end products of many catabolic processes are fed into the cycle to serve as fuels. Oxaloacetate and a-ketoglutarate are, for example, produced from asparate and glutamate, respectively, when proteins in the diet are degraded. Under other circumstances, intermediates are drawn out of the cycle to be used as precursors in a variety of biosynthetic pathways.

The pathway used in modern organisms is the product of evolution, much of which occurred before the advent of aerobic organisms. It does not necessarily represent the shortest pathway from acetate to CO₂, but is rather the pathway that has conferred the greatest selective advantage on its possessors throughout evolution. Early anaerobes very probably used some of the reactions of the citric acid cycle in linear biosynthetic processes. In fact, there are modern anaerobic microorganisms in which an incomplete citric acid cycle serves as a source, not of energy, but of biosynthetic precursors (Fig. 15-11). These organisms use the first three reactions of the citric acid cycle to make α-ketoglutarate, but they lack α-ketoglutarate dehydrogenase and therefore cannot carry out the complete set of citric acid cycle reactions. They do have the four enzymes that catalyze the reversible conversion of oxaloacetate into succinyl-CoA (Fig. 15-11), and these anaerobes make malate, fumarate, succinate, and succinyl-CoA from oxaloacetate in a reversal of the "normal" (oxidative) direction of flow through the cycle.

With the evolution of cyanobacteria that produced O₂ from water, the earth's atmosphere became aerobic and there was selective pressure on organisms to develop aerobic metabolism, which, as we have seen, is much more efficient than anaerobic fermentation.

Figure 15-11 The noncyclic reactions (blue arrows) that provide biosynthetic precursors in anaerobically growing bacteria. These cells lack α-ketoglutarate dehydrogenase and therefore cannot carry out the complete citric acid cycle, which normally follows the direction shown with gray arrows. α-Ketoglutarate and succinyl-CoA serve as precursors in a variety of biosynthetic reactions (see Fig. 15-12 ).

Citric Acid Cycle Components Are Important Biosynthetic Intermediates

In aerobic organisms, the citric acid cycle is an amphibolic pathway (i.e., it serves in both catabolic and anabolic processes). It not only functions in the oxidative catabolism of carbohydrates, fatty acids, and amino acids, but also provides precursors for many biosynthetic pathways (Fig. 15-12), as in anaerobic ancestors. By the action of several important auxiliary enzymes, certain intermediates of the citric acid cycle, particularly α-ketoglutarate and oxaloacetate, can be removed from the cycle to serve as precursors of amino acids. Aspartate and glutamate have the same carbon skeletons as oxaloacetate and α-ketoglutarate, respectively, and are synthesized from them by simple transamination (Chapter 21). Through aspartate and glutamate the carbons of oxaloacetate and α-ketoglutarate are used to build other amino acids as well as purine and pyrimidine nucleotides. We will see in Chapter 19 how oxaloacetate is converted into glucose in the process of gluconeogenesis. Succinyl-CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups, which serve as oxygen carriers (in hemoglobin and myoglobin) and electron carriers (in cytochromes).

Given the number of biosynthetic products derived from citric acid cycle intermediates, this cycle clearly serves a critical role apart from its function in energy-yielding metabolism.

Figure 15-12 Intermediates of the citric acid cycle are drawn off as precursors in many biosynthetic pathways, yielding the products in the shaded areas. Shown in red are four anaplerotic reactions that replenish depleted intermediates of the citric acid cycle (see Table 15-3).

Anaplerotic Reactions Replenish Citric Acid Cycle Intermediates

When intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, the resulting decrease in the concentration of these intermediates would be expected to slow the flux through the citric acid cycle. However, the intermediates can be replenished by anaplerotic reactions (Fig. 15-12; Table 15-3). Under normal circumstances the reactions by which the cycle intermediates are drained away and those by which they are replenished are in dynamic balance, so that the concentrations of the citric acid cycle intermediates remain almost constant.

In animal tissues one important anaplerotic reaction is the reversible carboxylation of pyruvate by CO₂ to form oxaloacetate, catalyzed by pyruvate carboxylase (Table 15-3). The three other anaplerotic reactions shown in Table 15-3 also serve, in various tissues and organisms, to convert either pyruvate or phosphoenolpyruvate to oxaloacetate. When the citric acid cycle is deficient in oxaloacetate or any of the other intermediates, pyruvate is carboxylated to produce more oxaloacetate. The enzymatic addition of a carboxyl group to the pyruvate molecule requires energy, which is supplied by ATP. Because the standard free-energy change of the overall reaction is very small, we can conclude that the free energy required to attach a carboxyl group to pyruvate is about equal to the free energy available from ATP. The carboxylation of pyruvate also requires the vitamin biotin (Fig. 15-13a), which is the prosthetic group of pyruvate carboxylase.

The pyruvate carboxylase reaction is the most important anaplerotic reaction in the liver and kidney of mammals. Other anaplerotic reactions are more important in other tissues and organisms (Table 15-3).

Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its positive allosteric modulator. Whenever acetyl-CoA, which is the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction.

The other anaplerotic reactions are also regulated to keep the level of intermediates high enough to support the activity of the citric acid cycle. Phosphoenolpyruvate carboxylase, for example, is activated by the glycolytic intermediate fructose-1,6-bisphosphate, the level of which rises when the citric acid cycle operates too slowly to process the pyruvate generated by glycolysis.

Biotin Carries O₂ Groups

Biotin plays a key role in many carboxylation reactions. This vitamin is a specialized carrier of one-carbon groups in their most oxidized form: CO₂. (The transfer of one-carbon groups in more reduced forms is mediated by other cofactors, notably tetrahydrofolate and S-adenosylmethionine, as described in Chapter 17.) Carboxyl groups are attached to biotin at the ureido group within the biotin ring system (Fig. 1513a).

Pyruvate carboxylase is composed of four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage between its valerate side chain and the e-amino group of a specific Lys residue in the enzyme active site (Fig. 15-13b); this biotinyllysine is called biocytin. Carboxylation of pyruvate proceeds in two steps; first, a carboxyl group derived from HCO₃^- is attached to biotin, then the carboxyl group is transferred to pyruvate to form oxaloacetate. These two steps occur at separate active sites; the long flexible arm of biocytin permits the transfer of activated carboxyl groups from the first active site to the second, much as the long lipoyllysyl arm of E₂ functions in the pyruvate dehydrogenase complex.

Biotin is a vitamin required in the human diet; it is abundant in many foods and is synthesized by intestinal bacteria. Deficiency diseases are rare and are generally observed only when large quantities of raw eggs are consumed. Egg whites contain a large amount of the protein avidin (M_r 70,000), which binds to biotin very tightly and prevents its absorption in the intestine. The avidin in egg whites may be a defense mechanism, inhibiting the growth of bacteria. When eggs are cooked, avidin is denatured (and thereby inactivated) along with all other egg white proteins.