Oxidation of Pyruvate and the TCA Cycle
Overview of Pyruvate Metabolism and the TCA Cycle
Under appropriate conditions, pyruvate can be further oxidized. One of the most studied oxidation reactions involving pyruvate is a two-part reaction involving NAD+ and a molecule called co-enzyme A, often abbreviated as "CoA". This reaction oxidizes pyruvate, leads to a loss of one carbon via decarboxylation, and creates a new molecule called acetyl-CoA. The resulting acetyl-CoA can enter several pathways for the biosynthesis of larger molecules or it can flow into another pathway of central metabolism called the Citric Acid Cycle, sometimes also called the Krebs Cycle, or Tricarboxylic Acid (TCA) Cycle. Here, the remaining two carbons in the acetyl group can either be further oxidized or serve again as precursors for the construction of various other molecules. We discuss these scenarios below.
The different fates of pyruvate and other end products of glycolysis
The glycolysis module left off with the end-products of glycolysis: 2 pyruvate molecules, 2 ATPs and 2 NADH molecules. This module and the module on fermentation explore what the cell can do with the pyruvate, ATP and NADH that were generated.
The fates of ATP and NADH
ATP can be used for or coupled to a variety of cellular functions including biosynthesis, transport, replication, etc. We will see many such examples throughout the course.
What to do with the NADH however, depends on the conditions under which the cell is growing. Sometimes, the cell will opt to recycle NADH rapidly back into NAD+. This occurs through a process called fermentation. This process returnsthe electrons initially taken from the glucose derivatives to more downstream products through another red/ox transfer (described in more detail in the module on fermentation). Alternatively, NADH can recycle back into NAD+ by donating electrons to something known as an electron transport chain (we cover this in the module on respiration and electron transport).
The fate of cellular pyruvate
- Pyruvate can be a terminal electron acceptor (either directly or indirectly) in fermentation reactions and we discuss this in the fermentation module.
- Pyruvate can be secreted from the cell as a waste product.
- Pyruvate can be further oxidized to extract more free energy from this fuel.
- Pyruvate can serve as a valuable intermediate compound linking some core carbon processing metabolic pathways
The further oxidation of pyruvate
In respiring bacteria and archaea, the pyruvate is further oxidized in the cytoplasm. In aerobically respiring eukaryotic cells, cells transport the pyruvate molecules produced at the end of glycolysis into mitochondria. These sites of cellular respiration house oxygen consuming electron transport chains (ETC in the module on respiration and electron transport). Organisms from all three domains of life share similar mechanisms to further oxidize the pyruvate to CO2. First pyruvate is decarboxylated and covalently linked to co-enzyme A via a thioester linkage to form the molecule known as acetyl-CoA. While acetyl-CoA can feed into multiple other biochemical pathways, we now consider its role in feeding the circular pathway known as the Tricarboxylic Acid Cycle, also referred to as the TCA cycle, the Citric Acid Cycle or the Krebs Cycle. We detail this process below.
Conversion of Pyruvate into Acetyl-CoA
In a multi-step reaction catalyzed by the enzyme pyruvate dehydrogenase, pyruvate is oxidized by NAD+, decarboxylated, and covalently linked to a molecule of co-enzyme A via a thioester bond. The release of the carbon dioxide is important here, this reaction often results in a loss of mass from the cell as the CO2 will diffuse or be transported out of the cell and become a waste product. In addition, cells reduce one molecule of NAD+ to NADH during this process per molecule of pyruvate oxidized. Remember: there are two pyruvate molecules produced at the end of glycolysis for every molecule of glucose metabolized; thus, if both pyruvate molecules are oxidized to acetyl-CoA two of the original six carbons will have converted to waste.
Figure 1. Upon entering the mitochondrial matrix, a multi-enzyme complex converts pyruvate into acetyl CoA. In the process, carbon dioxide is released and one molecule of NADH is formed.
In the presence of a suitable terminal electron acceptor, acetyl CoA delivers (exchanges a bond) its acetyl group to a four-carbon molecule, oxaloacetate, to form citrate (designated the first compound in the cycle). This cycle is called by different names: the citric acid cycle (for the first intermediate formed—citric acid, or citrate), the TCA cycle (since 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.
The Tricarboxcylic Acid (TCA) Cycle
In bacteria and archaea reactions in the TCA cycle typically happen in the cytosol. In eukaryotes, the TCA cycle takes place in the matrix of mitochondria. Almost all (but not all) of the enzymes of the TCA cycle are water soluble (not in the membrane), with the single exception of the enzyme succinate dehydrogenase, which is embedded in the inner membrane of the mitochondrion (in eukaryotes). Unlike glycolysis, the TCA 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 red/ox, dehydration, hydration, and decarboxylation reactions that produce two carbon dioxide molecules, one ATP, and reduced forms of NADH and FADH2.
Figure 2. In the TCA cycle, the acetyl group from acetyl CoA attaches to a four-carbon oxaloacetate molecule to form a six-carbon citrate molecule. Through a series of steps, citrate is oxidized, releasing two carbon dioxide molecules for each acetyl group fed into the cycle. In the process, three NAD+ molecules are reduced to NADH, one FAD+ molecule is reduced to FADH2, and one ATP or GTP (depending on the cell type) is produced (by substrate-level phosphorylation). Because the final product of the TCA cycle is also the first reactant, the cycle runs continuously in the presence of sufficient reactants.
Attribution: “Yikrazuul”/Wikimedia Commons (modified)
We are explicitly referring to eukaryotes, bacteria and archaea when we discuss the location of the TCA cycle because many beginning students of biology only associate the TCA cycle with mitochondria. Yes, the TCA cycle occurs in the mitochondria of eukaryotic cells. However, this pathway is not exclusive to eukaryotes; it occurs in bacteria and archaea too!
Steps in the TCA Cycle
The first step of the cycle is a condensation reaction involving the two-carbon acetyl group of acetyl-CoA with one four-carbon molecule of oxaloacetate. The products of this reaction are the six-carbon molecule citrate and free co-enzyme A. This step is considered irreversible because it is so highly exergonic. ATP concentration controls the rate of this reaction through negative feedback. If ATP levels increase, the rate of this reaction decreases. If ATP is in short supply, the rate increases. If not already, the reason will become clear shortly.
In step two, citrate loses one water molecule and gains another as citrate converts into its isomer, isocitrate.
In step three, isocitrate is oxidized by NAD+ and decarboxylated. Keep track of the carbons! This carbon now more than likely leaves the cell as waste and is no longer available for building new biomolecules. The oxidation of isocitrate therefore produces a five-carbon molecule, α-ketoglutarate, a molecule of CO2 and NADH. This step is also regulated by negative feedback from ATP and NADH, and via positive feedback from ADP.
Step 4 is catalyzed by the enzyme succinate dehydrogenase. Here, α-ketoglutarate is further oxidized by NAD+. This oxidation again leads to a decarboxylation and thus the loss of another carbon as waste. So far two carbons have come into the cycle from acetyl-CoA and two have left as CO2. At this stage, there is no net gain of carbons assimilated from the glucose molecules that are oxidized to this stage of metabolism. Unlike the previous step however succinate dehydrogenase - like pyruvate dehydrogenase before it - couples the free energy of the exergonic red/ox and decarboxylation reaction to drive the formation of a thioester bond between the substrate co-enzyme A and succinate (what is left after the decarboxylation). Succinate dehydrogenase is regulated by feedback inhibition of ATP, succinyl-CoA, and NADH.
Possible NB Discussion Point
We have seen several steps in this and other pathways that are regulated by allosteric feedback mechanisms. Why is it so important to be able to regulate cellular processes in the context of metabolism? Is there something(s) common to these regulated steps in the TCA cycle? Why might these steps be good steps to regulate in particular?
In step five, a substrate level phosphorylation event occurs. Here, an inorganic phosphate (Pi) is added to GDP or ADP to form GTP (an ATP equivalent for our purposes) or ATP. The energy that drives this substrate level phosphorylation event comes from the hydrolysis of the CoA molecule from succinyl~CoA to form succinate. Why is GTP or ATP produced? In animal cells there are two isoenzymes (different forms of an enzyme that carries out the same reaction), for this step, depending upon the animal tissue in which we find those cells. We find one isozyme in tissues that use large amounts of ATP, such as heart and skeletal muscle. This isozyme produces ATP. We find the second isozyme of the enzyme in tissues that have many anabolic pathways, such as liver. This isozyme produces GTP. GTP is energetically equivalent to ATP; However, its use is more restricted. In particular, the process of protein synthesis primarily uses GTP. Most bacterial systems produce GTP in this reaction.
Step six is another red/ox reactions in which succinate is oxidized by FAD+ into fumarate. Two hydrogen atoms are transferred to FAD+, producing FADH2. The difference in reduction potential between the fumarate/succinate and NAD+/NADH half reactions does not make NAD+ a suitable reagent for oxidizing succinate with NAD+ under cellular conditions. However, the difference in reduction potential with the FAD+/FADH2 half reaction is adequate to oxidize succinate and reduce FAD+. Unlike NAD+, FAD+ remains attached to the enzyme and transfers electrons to the electron transport chain directly. A cell makes this process possible by localizing the enzyme catalyzing this step inside the inner membrane of the mitochondrion or plasma membrane (depending on whether the organism in question is eukaryotic).
Water is added to fumarate during step seven, and malate is produced. The last step in the citric acid cycle regenerates oxaloacetate by oxidizing malate with NAD+. Another molecule of NADH is produced.
Note that this process (oxidation of pyruvate to Acetyl-CoA followed by one "turn" of the TCA cycle) completely oxidizes 1 molecule of pyruvate, a 3 carbon organic acid, to 3 molecules of CO2. Overall, 4 molecules of NADH, 1 molecule of FADH2, and 1 molecule of GTP (or ATP) are also produced. For respiring organisms this is a significant mode of energy extraction, since each molecule of NADH and FAD2 can feed directly into the electron transport chain, and as we will soon see, the subsequent red/ox reactions that are driven by this process will indirectly power the synthesis of ATP. The discussion so far suggests that the TCA cycle is primarily an energy extracting pathway; evolved to extract or convert as much potential energy from organic molecules to a form that cells can use, ATP (or the equivalent) or an energized membrane. However - and let us not forget - the other important outcome of evolving this pathway is the ability to produce several precursors or substrate molecules necessary for various catabolic reactions (this pathway provides some early building blocks to make bigger molecules). As we will discuss below, there is a strong link between carbon metabolism and energy metabolism.
Connections to Carbon Flow
One hypothesis that we have explored in this reading and in class is the idea that "central metabolism" evolved to generate carbon precursors for catabolic reactions. Our hypothesis also states that as cells evolved, these reactions became linked into pathways: glycolysis and the TCA cycle, to maximize their effectiveness for the cell. We can postulate that a side benefit to evolving this metabolic pathway was the generation of NADH from the complete oxidation of glucose - we saw the beginning of this idea when we discussed fermentation. We have already discussed how glycolysis not only provides ATP from substrate level phosphorylation but also yields a net of 2 NADH molecules and 6 essential precursors: glucose-6-P, fructose-6-P, 3-phosphoglycerate, phosphoenolpyruvate, and pyruvate. While ATP can be used by the cell directly as an energy source, NADH poses a problem and must be recycled back into NAD+, to keep the pathway in balance. As we see in the fermentation module, the most ancient way cells deal with this problem is to use fermentation reactions to regenerate NAD+.
During the process of pyruvate oxidation via the TCA cycle, 4 additional essential precursors are formed: acetyl~CoA, α-ketoglutarate, oxaloacetate, and succinyl~CoA. Three molecules of CO2 are lost, and this represents a net loss of mass for the cell. These precursors, however, are substrates for a variety of catabolic reactions including the production of amino acids, fatty acids, and various co-factors, such as heme. This means that the rate of reactions through the TCA cycle will be sensitive to the concentrations of each metabolic intermediate (more on the thermodynamics in class). A metabolic intermediate is a compound that is produced by one reaction (a product) and then acts as a substrate for the next reaction. This also means that metabolic intermediates, in particular the 4 essential precursors, can be removed for catabolic reactions, if there is a demand, changing the thermodynamics of the cycle.
Possible NB Discussion Point
Some of the TCA cycle intermediates, particularly glutamate and succinyl-CoA, are diverted away from the TCA cycle to other reactions. Unless something is done about it, this can leave the TCA cycle with too few intermediates to function effectively. Either through your own imagination or external study (hint: search "anapleurotic reactions" with a search engine) describe how this problem might be solved. What is required for this to happen? As a follow-up, you can also try to explain what you might need to run the TCA cycle backwards. Many organisms need to do this. What is required for this to happen and under what circumstances might it be helpful? Search "reductive Krebs Cycle" for leads.
Here are some additional links to videos and pages that you may find useful.
- Chemwiki TCA cycle - link down until key content corrections are made to the resource
Khan Academy Links
- Khan Academy TCA cycle - link down until key content corrections are made to the resource