9.22: Bis2A_Singer_Pyruvate_Oxidation_ and_ the_TCA_Cycle

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)

 

NOTE:
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

Step 1:

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.

Step 2:

In step two, citrate loses one water molecule and gains another as citrate converts into its isomer, isocitrate.

Step 3:

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:

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.

 

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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? 

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Step 5:

In step five, a substrate level phosphorylation event occurs. Here an inorganic phosphate (P_i) 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 type of 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 6:

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 or not the organism in question is eukaryotic).

Step 7:

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 in the process.

Summary

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.

 

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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. 

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Not all cells have a functional TCA cycle.

Since all cells require the ability of make these precursor molecules, one might expect that all organisms would have a fully functional TCA cycle. In fact, the cells of many organisms DO NOT have all the enzymes required to form a complete cycle - all cells, however, DO have the capability of making the 4 TCA cycle precursors noted in the previous paragraph. How can the cells make precursors and not have a full cycle? Remember that most of these reactions are freely reversible, so, if NAD⁺ is required to for the oxidation of pyruvate or acetyl~CoA, then the reverse reactions would require NADH. We often refer to this process as the reductive TCA cycle. To drive these reactions in reverse (regarding the direction discussed above) requires energy, in this case carried by ATP and NADH. If you get ATP and NADH driving a pathway one direction, driving it in reverse will require ATP and NADH as "inputs". So, organisms that do not have a full cycle can still make the 4 key metabolic precursors by using previously extracted energy and electrons (ATP and NADH) to drive some key steps in reverse.

Additional Links

Here are some additional links to videos and pages that you may find useful.

Chemwiki Links

• 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

 

Introduction into the pentose phosphate pathway (PPP)

Discussions of metabolism in most introductory biology courses focus on glycolysis (oxidation of glucose to pyruvate) and the TCA cycle (oxidation of pyruvate to acetyl-CoA and the eventual complete oxidation to CO₂). While these are important and universal metabolic pathways, many courses leave out the pentose phosphate pathway (PPP), also known as the hexose monophosphate shunt. In this class, we consider the PPP important for two key reasons.  First, it is the primary route for the formation of pentoses, the five-carbon sugar required for nucleotide biosynthesis and a variety of other essential cellular components. Second, redox reactions in the PPP generate NADPH, the main mobile electron donor used in anabolic (building) reactions. 

A note from the instructor

Like the modules on glycolysis and the TCA cycle, we do not expect students to memorize specific compound names or details of molecular structures in the pathway.   We provide those details in the reading so you can understand the transformations occurring in this pathway and refer back to them when needed. Rather than memorizing, focus instead on the mastering the assigned learning goals related to the PPP.  

Oxidative pentose phosphate pathway: a.k.a., the hexose monophosphate shunt

We call glycolysis, the TCA cycle and the pentose phosphate pathway central carbon metabolism. These three pathways (along with the reaction that converts pyruvate to acetyl-CoA) contain all the chemical precursors required by cells for the biosynthesis of nearly all other biomolecules. The PPP produces pentose phosphates (five-carbon sugars), eyrthrose-phosphate (a four-carbon sugar), and NADPH. The pentose phosphates are key precursors for nucleotide biosynthesis while NADPH serves as the main mobile electron donor for anabolic (building) reactions. The PPP also produces sedoheptulose-phosphate, an essential seven-carbon sugar used in the building of Gram-negative bacteria's outer cell membranes.

Below is a diagram of the pathway. The pathway involves several redox reactions and multiple molecular rearrangement that interconvert molecules of 3-, 4-, 5-, 6-, and 7-carbons. The pathway begins with the oxidation of Glucose-6-phosphate (G6P), a key intermediate of glycolysis, by the enzyme glucose-6-phosphate dehydrogenase (G6PDH). This enzyme oxidizes G6P through the coupled reduction of the electron carrier NADP+ to make NADPH.  Enzymes called transaldolases and transketalases are used to produce the intermediates within the pathway. The net result is oxidation and subsequent decarboxylation of glucose to form a pentose. The total reaction involves three glucose-6-phosphate (in green) molecules being oxidized to form three CO₂ molecules, one glyceraldehyde-phosphate (in red), and two hexose-phosphates (in red). In this cycle, the formed glyceradehyde-phosphate feeds into glycolysis and the two hexose-phosphates (e.g., glucose-phosphates) can recycle into the PPP or glycolysis.

Figure 1. The Pentose Phosphate Pathway (PPP).

The Pentose Phosphate Pathway diverts the glucose-6-phosphate from glycolysis, oxidizes the sugar to produce NADPH for anabolic reactions, creates ribose-5-phosphate for nucleotide biosynthesis, and other key 3, 4 and 7 carbon intermediates.  Two of the products, fructose-6-phosphate and glyceraldehyde-3-phosphate reenter glycolysis. Compound names are blue while enzyme names are red italic font.  
Attribution: Marc T. Facciotti 

As shown in the figure above, products of the pathway include glyceraldehyde-3-phosphate. This sugar can then be further oxidized via glycolysis. Fructose-6-phosphate that can reenter glycolysis and NADPH, a  reductant for many biosynthetic (anabolic) reactions is also made. In addition, the pathway provides a variety of intermediate sugar-phosphates that the cell requires, such as pentose-phosphates (for nucleotides and some amino acids), erythrose-phosphate (for amino acids) and sedohepulose-phosphate (for gram-negative bacteria).  The figure below illustrates the input-output relationship between PPP and the "top half" of glycolysis.

 

 

 

Figure 2. The relationship between glycolysis and PPP.

The Pentose Phosphate Pathway is shown as a "shunt" (alternative metabolic pathway) for glucose-6-phosphate.  
Attribution: Marc T. Facciotti