5.4 Oxidation of Pyruvate and the Citric Acid Cycle

Learning Objectives

By the end of this section, you will be able to:

  • Describe how pyruvate, the product of glycolysis, is prepared for entry into the citric acid cycle
  • Explain how a circular pathway, such as the citric acid cycle, fundamentally differs from a linear biochemical pathway, such as glycolysis
  • Describe the location of the citric acid cycle in the cell
  • Describe the products of the citric acid cycle

Overview of the Citric Acid Cycle

In eukaryotic cells, the pyruvate molecules produced at the end of glycolysis are transported into mitochondria, which are sites of cellular respiration. If oxygen is available, aerobic respiration will go forward. In mitochondria, pyruvate will be transformed into a two-carbon acetyl group (by removing a molecule of carbon dioxide) that will be picked up by a carrier compound called coenzyme A (CoA), which is made from vitamin B5, pantothenic acid. The resulting compound is called acetyl CoA (Figure 5.14). Acetyl CoA can be used in a variety of ways by the cell, but its major function is to deliver the acetyl group derived from pyruvate to the next pathway in glucose catabolism.

A graphic shows pyruvate becoming a two-carbon acetyl group by removing one molecule of carbon dioxide. The two-carbon acetyl group is picked up by coenzyme A to become acetyl CoA. The acetyl CoA then enters the citric acid cycle. Three NADH, one FADH2, one ATP, and two carbon dioxide molecules are produced during this cycle.
Figure 5.14 Pyruvate is converted into acetyl CoA before entering the citric acid cycle.

Like the conversion of pyruvate to acetyl CoA, the citric acid cycle in eukaryotic cells takes place in the matrix of the mitochondria. Almost all of the enzymes of the citric acid cycle are soluble, with the single exception of the enzyme succinate dehydrogenase, which is embedded in the inner membrane of the mitochondrion. Unlike glycolysis, the citric acid cycle is a closed loop: The last part of the pathway regenerates the compound used in the first step. The eight steps of the cycle are a series of redox, dehydration, hydration, and decarboxylation reactions that produces two carbon dioxide molecules, one ATP molecule (or an equivalent GTP), and reduced forms (NADH and FADH2) of NAD+ and FAD, important coenzymes in the cell. Part of this is considered an aerobic pathway (oxygen-requiring) because the NADH and FADH2 produced must transfer their electrons to the next pathway in the system, which will use oxygen. If oxygen is not present, this transfer does not occur. Note that the citric acid cycle produces very little ATP directly and does not directly consume oxygen.

The citric acid cycle is called by different names: the citric acid cycle (for the first intermediate formed—citric acid, or citrate—when acetate joins to the oxaloacetate), the TCA cycle (because citric acid or citrate and isocitrate are tricarboxylic acids), and the Krebs cycle, after Hans Krebs, who first identified the steps in the pathway in the 1930s in pigeon flight muscles.

Oxidation of Pyruvate

In order for pyruvate, the product of glycolysis, to enter the citric acid cycle, it must undergo several changes. The conversion is a three-step process (Figure 5.15).

Step 1. A carboxyl group is removed from pyruvate, releasing a molecule of carbon dioxide into the surrounding medium. This reaction creates a two-carbon hydroxyethyl group bound to the enzyme (pyruvate dehydrogenase). We should note that this is the first of the six carbons from the original glucose molecule to be removed. (This step proceeds twice because there are two pyruvate molecules produced at the end of glycolsis for every molecule of glucose metabolized anaerobically; thus, two of the six carbons will have been removed at the end of both steps.)

Step 2. The hydroxyethyl group is oxidized to an acetyl group, and the electrons are picked up by NAD+, forming NADH. The high-energy electrons from NADH will be used later to generate ATP.

Step 3. The enzyme-bound acetyl group is transferred to CoA, producing a molecule of acetyl CoA.

Figure 5.15 Pyruvate begins outside the mitochondrion, in the cytosol. When the pyruvate enters the mitochondrion through the pyruvate carrier protein, a carboxyl group is removed from pyruvate, releasing carbon dioxide. Then, a redox reaction forms acetate and NADH. The acetate is transferred to coenzyme A (CoA-SH) to form acetyl CoA.

The Citric Acid Cycle

The intermediates of the citric acid cycle are in Figure 5.16.

Step 1. Prior to the first step, a transitional phase occurs during which pyruvic acid is converted to acetyl CoA. Then, the first step of the cycle begins: This condensation step combines the two-carbon acetyl group with a four-carbon oxaloacetate molecule to form a six-carbon molecule of citrate. CoA is bound to a sulfhydryl group (-SH) and diffuses away to eventually combine with another acetyl group. This step is irreversible because it is highly exergonic. The rate of this reaction is controlled by negative feedback and the amount of ATP available. If ATP levels increase, the rate of this reaction decreases. If ATP is in short supply, the rate increases.

Step 2. In step two, citrate loses one water molecule and gains another as citrate is converted into its isomer, isocitrate.

Step 3. In step three, isocitrate is oxidized, producing a five-carbon molecule, α-ketoglutarate, along with a molecule of CO2 and two electrons, which reduce NAD+ to NADH. This step is also regulated by negative feedback from ATP and NADH and a positive effect of ADP.

Step 4. Steps three and four are both oxidation and decarboxylation steps, which as we have seen, release electrons that reduce NAD+ to NADH and release carboxyl groups that form CO2 molecules. Alpha-ketoglutarate is the product of step three, and a succinyl group is the product of step four. CoA binds with the succinyl group to form succinyl CoA. The enzyme that catalyzes step four is regulated by feedback inhibition of ATP, succinyl CoA, and NADH.

Step 5. In step five, a carboxyl group is substituted for coenzyme A, and a high-energy bond is formed. This energy is used in substrate-level phosphorylation (during the conversion of the succinyl group to succinate) to form either guanine triphosphate (GTP) or ATP. There are two forms of the enzyme, called isoenzymes, for this step, depending upon the type of animal tissue in which they are found. One form is found in tissues that use large amounts of ATP, such as heart and skeletal muscle. This form produces ATP. The second form of the enzyme is found in tissues that have a high number of anabolic pathways, such as liver. This form produces GTP. GTP is energetically equivalent to ATP; however, its use is more restricted. In particular, protein synthesis primarily uses GTP.

Step 6. Step six is a dehydration process that converts succinate into fumarate. Two hydrogen atoms are transferred to FAD, reducing it to FADH2. (Note: the energy contained in the electrons of these hydrogens is insufficient to reduce NAD+ but adequate to reduce FAD.) Unlike NADH, this carrier remains attached to the enzyme and transfers the electrons to the electron transport chain directly. This process is made possible by the localization of the enzyme catalyzing this step inside the inner membrane of the mitochondrion.

Step 7. Water is added by hydrolysis to fumarate during step seven, and malate is produced.

Step 8: The last step in the citric acid cycle regenerates oxaloacetate by oxidizing malate. Another molecule of NADH is then produced in the process.

Figure 5.16 This illustration shows the eight steps of the citric acid cycle. In the first step, the acetyl group from acetyl CoA is transferred to a four-carbon oxaloacetate molecule to form a six-carbon citrate molecule. In the second step, citrate is rearranged to form isocitrate. In the third step, isocitrate is oxidized to alpha-ketoglutarate. In the process, one NADH is formed from NAD+; and one carbon dioxide is released. In the fourth step, alpha-ketoglutarate is oxidized CoA is added, forming succinyl CoA. In the process, another NADH is formed and another carbon dioxide is released. In the fifth step, CoA is released from succinyl CoA, forming succinate. In the process, one GTP is formed, which is later converted into ATP. In the sixth step, succinate is oxidized to fumarate, and one FAD is reduced to FADH2. In the seventh step, fumarate is converted into malate. In the eighth step, malate is oxidized to oxaloacetate, and another NADH is formed. (Credit: modification of work by “Yikrazuul”/Wikimedia Commons)

Products of the Citric Acid Cycle

Two carbon atoms come into the citric acid cycle from each acetyl group, representing four out of the six carbons of one glucose molecule. Two carbon dioxide molecules are released on each turn of the cycle; however, these do not necessarily contain the most recently added carbon atoms. The two acetyl carbon atoms will eventually be released on later turns of the cycle; thus, all six carbon atoms from the original glucose molecule are eventually incorporated into carbon dioxide. Each turn of the cycle forms three NADH molecules and one FADH2 molecule. These carriers will connect with the last portion of aerobic respiration, the electron transport chain, to produce ATP molecules. One GTP or ATP is also made in each cycle. Several of the intermediate compounds in the citric acid cycle can be used in synthesizing nonessential amino acids; therefore, the cycle is amphibolic (both catabolic and anabolic).

LINK TO LEARNING

Click through each step of the citric acid cycle here.

Section Summary

The citric acid cycle is a series of chemical reactions that removes high-energy electrons and uses them in the electron transport chain to generate ATP. One molecule of ATP (or an equivalent) is produced per each turn of the cycle.

Exercises

Glossary

acetyl CoA: the combination of an acetyl group derived from pyruvic acid and coenzyme A which is made from pantothenic acid (a B-group vitamin)

ATP synthase: a membrane-embedded protein complex that regenerates ATP from ADP with energy from protons diffusing through it

citric acid cycle: a series of enzyme-catalyzed chemical reactions of central importance in all living cells that harvests the energy in carbon-carbon bonds of sugar molecules to generate ATP; the citric acid cycle is an aerobic metabolic pathway because it requires oxygen in later reactions to proceed

electron transport chain: a series of four large, multi-protein complexes embedded in the inner mitochondrial membrane that accepts electrons from donor compounds and harvests energy from a series of chemical reactions to generate a hydrogen ion gradient across the membrane

Media Attribution

  • Figure 5.16 modification of work by “Yikrazuul”/Wikimedia Commons

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Introduction to Biology Copyright © 2023 by Natasha Ramroop Singh, PhD is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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