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.

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.

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 formedgives some details of this episode in the scientific history of the cycle.

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. 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. 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  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 (Mr 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.