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Chapter: Biochemistry: The Citric Acid Cycle

The Individual Reactions of the Citric Acid Cycle

The reactions of the citric acid cycle proper and the enzymes that catalyze them are listed in Table 19.1. We shall now discuss each of these reactions in turn.

The Individual Reactions of the Citric Acid Cycle

The reactions of the citric acid cycle proper and the enzymes that catalyze them are listed in Table 19.1. We shall now discuss each of these reactions in turn.

Step 1.Formation of CitrateThe first step of the citric acid cycle is thereaction of acetyl-CoA and oxaloacetate to form citrate and CoA-SH. This reaction is called a condensation because a new carbon–carbon bond is formed. The condensation reaction of acetyl-CoA and oxaloacetate to form citryl-CoA takes place in the first stage of the reaction. The condensation is followed by the hydrolysis of citryl-CoA to give citrate and CoA-SH.

The reaction is catalyzed by the enzyme citrate synthase, originally called “condensing enzyme.” A synthase is an enzyme that makes a new covalent bond during the reaction, but it does not require the direct input of ATP. It is an exergonic reaction ( ∆G°' = –32.8 kJ mol–1 = –7.8 kcal mol–1) because the hydrolysis of a thioester releases energy. Thioesters are considered high-energy compounds.

Step 2.Isomerization of Citrate to IsocitrateThe second reaction of the citricacid cycle, the one catalyzed by aconitase, is the isomerization of citrate to isocitrate. The enzyme requires Fe2+. One of the most interesting features of the reaction is that citrate, a symmetrical (achiral) compound, is converted to isocitrate, a chiral compound, a molecule that cannot be superimposed on its mirror image.

It is often possible for a chiral compound to have several different isomers. Isocitrate has four possible isomers, but only one of the four is produced by this reaction. Aconitase, the enzyme that catalyzes the conversion of citrate to isoci-trate, can select one end of the citrate molecule in preference to the other.

This type of behavior means that the enzyme can bind a symmetrical substrate in an unsymmetrical binding site. We mentioned that this possibility exists, and here we have an example of it. The enzyme forms an unsymmetrical three-point attachment to the citrate molecule (Figure 19.6). The reaction proceeds by removal of a water molecule from the citrate to produce cis-aconitate, and then water is added back to the cis-aconitate to give isocitrate.

The intermediate, cis-aconitate, remains bound to the enzyme during the course of the reaction. There is some evidence that the citrate is complexed to the Fe(II) in the active site of the enzyme in such a way that the citrate curls back on itself in a nearly circular conformation. Several authors have been unable to resist the temptation to call this situation the “ferrous wheel.”

Step 3.Formation of -Ketoglutarate and CO2—First OxidationThe thirdstep in the citric acid cycle is the oxidative decarboxylation of isocitrate to α-ketoglutarate and carbon dioxide. This reaction is the first of two oxidativedecarboxylations of the citric acid cycle; the enzyme that catalyzes it is isocitrate dehydrogenase. The reaction takes place in two steps (Figure 19.7).First, isocitrate is oxidized to oxalosuccinate, which remains bound to the enzyme. Then oxalosuccinate is decarboxylated, and the carbon dioxide and α-ketoglutarate are released.

This is the first of the reactions in which NADH is produced. One molecule of NADH is produced from NAD+ at this stage by the loss of two electrons in the oxidation. As we saw in our discussion of the pyruvate dehydrogenase com-plex, each NADH produced leads to the production of 2.5 ATP in later stages of aerobic metabolism. Recall also that there will be two NADH, equivalent to five ATP for each original molecule of glucose.

Step 4.Formation of Succinyl-CoA and CO2—Second OxidationThesecond oxidative decarboxylation takes place in Step 4 of the citric acid cycle, in which carbon dioxide and succinyl-CoA are formed from α-ketoglutarate and CoA.

This reaction is similar to the one in which acetyl-CoA is formed from pyruvate, with NADH produced from NAD+. Once again, each NADH eventually gives rise to 2.5 ATP, with five ATP from each original molecule of glucose.

The reaction occurs in several stages and is catalyzed by an enzyme system called the α-ketoglutarate dehydrogenase complex, which is very similar to the pyruvate dehydrogenase complex. Each of these multienzyme systems consists of three enzymes that catalyze the overall reaction. The reaction takes place in several steps, and there is again a requirement for thiamine pyrophosphate (TPP), FAD, lipoic acid, and Mg2+. This reaction is highly exergonic ( ∆G°' = –33.4 kJ mol–1 = –8.0 kcal mol–1), as is the one catalyzed by pyruvate dehydrogenase.

At this point, two molecules of CO2 have been produced by the oxidative decarboxylations of the citric acid cycle. Removal of the CO2 makes the citric acid cycle irreversible in vivo, although in vitro each separate reaction is revers-ible. One might suspect that the two molecules of CO2 arise from the two car-bon atoms of acetyl-CoA. Labeling studies have shown that this is not the case, but a full discussion of this point is beyond the scope of this text.

The two CO2 arise from carbon atoms that were part of the oxaloacetate with which the acetyl group condensed. The carbons of this acetyl group are incorporated into the oxaloacetate that will be regenerated for the next round of the cycle. The release of the CO2 molecules has a profound influence on mammalian physiology, as will be discussed later. We should also mention that the α-ketoglutarate dehydrogenase complex reaction is the third one in which we have encountered an enzyme that requires TPP.

Step 5.Formation of SuccinateIn the next step of the cycle, the thioester bondof succinyl-CoA is hydrolyzed to produce succinate and CoA-SH; an accompanying reaction is the phosphorylation of GDP to GTP. The whole reaction is catalyzed by the enzyme succinyl-CoA synthetase. A synthetase is an enzyme that creates a new covalent bond and requires the direct input of energy from a high-energy phosphate. Recall that we met a synthase (citrate synthase) earlier. The difference between a synthase and a synthetase is that a synthase does not require energy from phosphate-bond hydrolysis, whereas a synthetase does. In the reaction mechanism, a phosphate group covalently bonded to the enzyme is directly transferred to the GDP. The phosphorylation of GDP to GTP is endergonic, as is the corresponding ADP-to-ATP reaction ( ∆G°' = 30.5 kJ mol–1 = 7.3 kcal mol–1).

The energy required for the phosphorylation of GDP to GTP is provided by the hydrolysis of succinyl-CoA to produce succinate and CoA. The free energy of hydrolysis ( ∆G°') of succinyl-CoA is –33.4 kJ mol–1 (–8.0 kcal mol–1). The overall reaction is slightly exergonic ( ∆G°' = –3.3 kJ mol–1 = –0.8 kcal mol–1) and, as a result, does not contribute greatly to the overall production of energy by the mitochondrion. Note that the name of the enzyme describes the reverse reaction. Succinyl-CoA synthetase would produce succinyl-CoA while spending an ATP or another high-energy phosphate. This reaction is the opposite of that.

The enzyme nucleosidediphosphate kinase catalyzes the transfer of a phos-phate group from GTP to ADP to give GDP and ATP.


This reaction step is called substrate-level phosphorylation to distinguish it from the type of reaction for production of ATP that is coupled to the electron transport chain. The production of ATP in this reaction is the only place in the citric acid cycle in which chemical energy in the form of ATP is made available to the cell. Except for this reaction, the generation of ATP characteristic of aerobic metabolism is associated with the electron transport chain. About 30 to 32 molecules of ATP can be obtained from the oxidation of a single molecule of glucose by the combination of anaerobic and aerobic oxidation, compared with only two molecules of ATP produced by anaerobic glycolysis alone. The combined reactions that occur in mitochondria are of great importance to aerobic organisms.

In the next three steps in the citric acid cycle (Steps 6 through 8), the four-carbon succinate ion is converted to oxaloacetate ion to complete the cycle.

Step 6.Formation of Fumarate—FAD-Linked OxidationSuccinate isoxidized to fumarate, a reaction that is catalyzed by the enzyme succinatedehydrogenase. This enzyme is an integral protein of the inner mitochondrialmembrane. The other individual enzymes of the citric acid cycle are in the mitochondrial matrix. The electron acceptor, which is FAD rather than NAD+, is covalently bonded to the enzyme; succinate dehydrogenase is also called a flavoprotein because of the presence of FAD with its flavin moiety. In the succinate dehydrogenase reaction, FAD is reduced to FADH2 and succinate is oxidized to fumarate.

The overall reaction is

Succinate + E-FAD - > Fumarate + E-FADH2

The E-FAD and E-FADH2 in the equation indicate that the electron accep-tor is covalently bonded to the enzyme. The FADH2 group passes electrons on to the electron transport chain, and eventually to oxygen, and gives rise to 1.5 ATP, rather than 2.5, as is the case with NADH.

Succinate dehydrogenase contains iron atoms but does not contain a heme group; it is referred to as a nonheme iron protein or an iron-sulfur protein. The latter name refers to the fact that the protein contains several clusters that con-sist of four atoms each of iron and of sulfur.

Step 7.Formation ofL-MalateIn Step 7, which is catalyzed by the enzymefumarase, water is added across the double bond of fumarate in a hydrationreaction to give malate. Again, there is stereospecificity in the reaction. Malate has two enantiomers, L- and D-malate, but only L-malate is produced.

Step 8.Regeneration of Oxaloacetate—Final Oxidation StepMalateis oxidized to oxaloacetate, and another molecule of NAD+is reduced to NADH. This reaction is catalyzed by the enzyme malate dehydrogenase. The oxaloacetate can then react with another molecule of acetyl-CoA to start another round of the cycle.

The oxidation of pyruvate by the pyruvate dehydrogenase complex and the citric acid cycle results in the production of three molecules of CO2. As a result of these oxidation reactions, one molecule of GDP is phosphorylated to GTP, one molecule of FAD is reduced to FADH2, and four molecules of NAD+ are reduced to NADH. Of the four molecules of NADH produced, three come from the citric acid cycle, and one comes from the reaction of the pyruvate dehydrogenase complex. The overall stoichiometry of the oxidation reactions is the sum of the pyruvate dehydrogenase reaction and the citric acid cycle. Note that only one high-energy phosphate, GTP, is produced directly from the citric acid cycle, but many more ATP will arise from reoxidation of NADH and FADH2.

Pyruvate dehydrogenase complex:

Pyruvate + CoA-SH + NAD+ - > Acetyl-CoA + NADH + CO2 + H+

Citric acid cycle:

Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O ->

2CO2 + CoA-SH + 3NADH + 3H+ + FADH2 + GTP

Overall reaction:

Pyruvate + 4NAD+ + FAD + GDP + Pi + 2H2O  ->

3CO2 + 4NADH + FADH2 + GTP + 4H+

Eventual ATP production per pyruvate:

4NADH - > 10ATP (2.5ATP for each NADH)

1FADH - > 1.5ATP (1.5ATP for each FADH2)

1GTP - > 1ATP

Total 12.5 ATP per pyruvate or 25 ATP per glucose

There were also two ATP produced per glucose in glycolysis and two NADH, which will give rise to another five ATP (seven more ATP total).

At this point, we would do well to recapitulate what we have said about the citric acid cycle (see Figure 19.3). When studying a pathway such as this, we might learn many details but also be able to see the big picture. The entire pathway is shown with the enzyme names outside the circle. The most impor-tant reactions can be identified by those that have important cofactors (NADH, FADH2, GTP). Also important are the steps where CO2 is given off.

These important reactions also play a large role in the cycle’s contribution to our metabolism. One purpose of the cycle is to produce energy. It does that by producing GTP directly and by producing reduced electron carriers (NADH and FADH2). The three decarboxylations mean that for every three carbons entering as pyruvate, three carbons are effectively lost during the cycle, a fact that has many implications to our metabolism.


In the citric acid cycle and the pyruvate dehydrogenase reaction, one molecule of pyruvate is oxidized to three molecules of carbon dioxide as a result of oxidative decarboxylations

The oxidations are accompanied by reductions. Four NAD+ are reduced to NADH, and one FAD to FADH2; in addition, one GDP is phosphory-lated to GTP.


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