Introduction

In eukaryotic cells, the pyruvate molecules produced at the end of glycolysis are transported into the mitochondria, which are the sites of cellular respiration. There, pyruvate is transformed into an acetyl group that will be picked up and activated by a carrier compound called coenzyme A (CoA), which is derived from vitamin B5. The resulting compound is called acetyl CoA. 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 stage of the pathway in glucose catabolism.

Acetyl CoA is the primary reactant in the Citric Acid Cycle. By the end of these two processes, all 6 carbons that originally made up glucose are fully oxidized and released as CO₂ gas.

Pyruvate Processing

In order for pyruvate, the product of glycolysis, to enter the next pathway, it must undergo several changes. Pyruvate processing (also called pyruvate oxidation) occurs in the matrix of the mitochondria. The conversion is a three-step process (Figure 27.1).

Step 1: A carbon on pyruvate is oxidized and released as a molecule of carbon dioxide. 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 glycolysis for every molecule of glucose; thus, two of the six original 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.

Note that during the second stage of glucose metabolism, whenever a carbon atom is removed, it is bound to two oxygen atoms, producing carbon dioxide, one of the major end products of cellular respiration.

Thus, pyruvate processing converts two 3-carbon pyruvate molecules into two 2-carbon Acetyl Co-A molecules. The two missing carbons were fully oxidized and released as CO₂. The process also produced two reduced NADH molecules.

The Citric Acid Cycle

In the presence of oxygen, acetyl CoA delivers its acetyl (2 carbon, 2-C) group to a 4 carbon (4-C) molecule, oxaloacetate, to form citrate, a six-carbon molecule with three carboxyl groups; this pathway will harvest the remainder of the extractable energy from what began as a glucose molecule and release the remaining four CO₂ molecules. This single pathway 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. For simplicity, we will call it the Citric Acid Cycle, but you should be aware that it goes by these other names as well so that you can recognize it as the same process when you see these other names elsewhere.

Like the conversion of pyruvate to acetyl CoA, the Citric Acid Cycle takes place in the matrix of 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 produce two carbon dioxide molecules, one GTP/ATP, and the reduced carriers NADH and FADH₂ (Figure 27.2). Note that the citric acid cycle produces very little ATP directly and does not directly consume oxygen. Both of these things will occur in the final step of cellular respiration, which we will cover in detail in the next chapter.

Steps in the Citric Acid Cycle

Step 1: This condensation step combines the two-carbon acetyl group from Acetyl Co-A 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. This reaction is an isomerization and catalyzed by an isomerase.

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 3, and a succinyl group is the product of step 4. CoA binds with the succinyl group to form succinyl CoA. The enzyme that catalyzes step 4 is regulated by feedback inhibition of ATP, succinyl CoA, and NADH.

Step 5: In step five, a phosphate 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. The last step in the citric acid cycle regenerates oxaloacetate by oxidizing malate. Another molecule of NADH is then produced in the process

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. Thus, all six carbon atoms from the original glucose molecule are eventually fully oxidized 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. Remember that there will be two turns of the Citric Acid Cycle for every molecule of glucose because there are two pyruvates, and therefore two Acetyl Co-A molecules, per glucose.

Acknowledgements

Adapted from Clark, M.A., Douglas, M., and Choi, J. (2018). Biology 2e. OpenStax. Retrieved from https://openstax.org/books/biology-2e/pages/1-introduction