2020_Spring_Bis2A_Facciotti_Lecture_14

Learning objectives associated with 2020_Spring_Bis2A_Facciotti_Lecture_14 Create an “energy story” explaining the functional value of pyruvate reduction (fermentation) to a cell and its importance in regenerating the NAD+ pool. Discuss at least two key fates for glucose other than its direct oxidation to pyruvate, highlighting the role of glycolytic intermediates in processes related to carbon flow (functions that are not associated with energy harvesting).    Identify coenzyme A and its functionally important thiol group by inspection of a molecular model. Distinguish between the molecular structures of  ATP, NAD+ and Coenzyme-A from representations of their molecular structures. Relate Coenzyme A's ability to form thioesters with the concepts of its function of carrying “activated” acyl groups. Explain the central role of Acetyl CoA in carbon metabolism including its formation from various sources (the oxidative decarboxylation of pyruvate, oxidation of fatty acids, and/or oxidative degradation of some amino acids) and as a carbon source in both the TCA cycle and lipid synthesis. Create an energy story explaining the functional value of pyruvate oxidation and its importance in generating building blocks for biomolecules. Create an energy story for each reaction in the TCA cycle. Describe and give an example of the catalytic role that a cyclic pathway can have on energy harvesting and the inter-conversion of molecule types. Given metabolic maps of glycolysis and TCA cycle, follow the flow of electrons from energy source to mobile carrier NADH and use a provided redox tower to quantitatively describe the energy transformations.

 

Fermentation and regeneration of NAD+

Section summary

This section discusses the process of fermentation. Due to the heavy emphasis in this course on central carbon metabolism, the discussion of fermentation understandably focuses on the fermentation of pyruvate. Nevertheless, some of the core principles that we cover in this section apply equally well to the fermentation of many other small molecules.

The "purpose" of fermentation

The oxidation of a variety of small organic compounds is a process that is utilized by many organisms to garner energy for cellular maintenance and growth. The oxidation of glucose via glycolysis is one such pathway. Several key steps in the oxidation of glucose to pyruvate involve the reduction of the electron/energy shuttle NAD⁺ to NADH. You were already asked to figure out what options the cell might reasonably have to reoxidize the NADH to NAD⁺ in order to avoid consuming the available pools of NAD⁺ and to thus avoid stopping glycolysis. Put differently, during glycolysis, cells can generate large amounts of NADH and slowly exhaust their supplies of NAD⁺. If glycolysis is to continue, the cell must find a way to regenerate NAD⁺, either by synthesis or by some form of recycling.

In the absence of any other process—that is, if we consider glycolysis alone—it is not immediately obvious what the cell might do. One choice is to try putting the electrons that were once stripped off of the glucose derivatives right back onto the downstream product, pyruvate, or one of its derivatives. We can generalize the process by describing it as the returning of electrons to the molecule that they were once removed, usually to restore pools of an oxidizing agent. This, in short, is fermentation. As we will discuss in a different section, the process of respiration can also regenerate the pools of NAD⁺ from NADH. Cells lacking respiratory chains or in conditions where using the respiratory chain is unfavorable may choose fermentation as an alternative mechanism for garnering energy from small molecules.

An example: lactic acid fermentation

An everyday example of a fermentation reaction is the reduction of pyruvate to lactate by the lactic acid fermentation reaction. This reaction should be familiar to you: it occurs in our muscles when we exert ourselves during exercise. When we exert ourselves, our muscles require large amounts of ATP to perform the work we are demanding of them. As the ATP is consumed, the muscle cells are unable to keep up with the demand for respiration, O₂ becomes limiting, and NADH accumulates. Cells need to get rid of the excess and regenerate NAD⁺, so pyruvate serves as an electron acceptor, generating lactate and oxidizing NADH to NAD⁺. Many bacteria use this pathway as a way to complete the NADH/NAD⁺ cycle. You may be familiar with this process from products like sauerkraut and yogurt. The chemical reaction of lactic acid fermentation is the following:

Pyruvate + NADH ↔ lactic acid + NAD⁺

 

Figure 1. Lactic acid fermentation converts pyruvate (a slightly oxidized carbon compound) to lactic acid. In the process, NADH is oxidized to form NAD⁺. Attribution: Marc T. Facciotti (original work)

Energy story for the fermentation of pyruvate to lactate

An example (if a bit lengthy) energy story for lactic acid fermentation is the following: 

The reactants are pyruvate, NADH, and a proton. The products are lactate and NAD⁺. The process of fermentation results in the reduction of pyruvate to form lactic acid and the oxidation of NADH to form NAD⁺. Electrons from NADH and a proton are used to reduce pyruvate into lactate. If we examine a table of standard reduction potential, we see under standard conditions that a transfer of electrons from NADH to pyruvate to form lactate is exergonic and thus thermodynamically spontaneous. The reduction and oxidation steps of the reaction are coupled and catalyzed by the enzyme lactate dehydrogenase.

A second example: alcohol fermentation

Another familiar fermentation process is alcohol fermentation, which produces ethanol, an alcohol. The alcohol fermentation reaction is the following:

Figure 2. Ethanol fermentation is a two-step process. Pyruvate (pyruvic acid) is first converted into carbon dioxide and acetaldehyde. The second step converts acetaldehyde to ethanol and oxidizes NADH to NAD⁺. Attribution: Marc T. Facciotti (original work)

In the first reaction, a carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas (some of you may be familiar with this as a key component of various beverages). The second reaction removes electrons from NADH, forming NAD⁺ and producing ethanol (another familiar compound—usually in the same beverage) from the acetaldehyde, which accepts the electrons.
 

Fermentation pathways are numerous

While the lactic acid fermentation and alcohol fermentation pathways described above are examples, there are many more reactions (too numerous to go over) that Nature has evolved to complete the NADH/NAD⁺ cycle. It is important that you understand the general concepts behind these reactions. In general, cells try to maintain a balance or constant ratio between NADH and NAD⁺; when this ratio becomes unbalanced, the cell compensates by modulating other reactions to compensate. The only requirement for a fermentation reaction is that it uses a small organic compound as an electron acceptor for NADH and regenerates NAD⁺. Other familiar fermentation reactions include ethanol fermentation (as in beer and bread), propionic fermentation (it's what makes the holes in Swiss cheese), and malolactic fermentation (it's what gives Chardonnay its more mellow flavor—the more conversion of malate to lactate, the softer the wine). In Figure 3, you can see a large variety of fermentation reactions that various bacteria use to reoxidize NADH to NAD⁺. All of these reactions start with pyruvate or a derivative of pyruvate metabolism, such as oxaloacetate or formate. Pyruvate is produced from the oxidation of sugars (glucose or ribose) or other small, reduced organic molecules. It should also be noted that other compounds can be used as fermentation substrates besides pyruvate and its derivatives. These include methane fermentation, sulfide fermentation, or the fermentation of nitrogenous compounds such as amino acids. You are not expected to memorize all of these pathways. You are, however, expected to recognize a pathway that returns electrons to products of the compounds that were originally oxidized to recycle the NAD⁺/NADH pool and to associate that process with fermentation.

Figure 3. This figure shows various fermentation pathways using pyruvate as the initial substrate. In the figure, pyruvate is reduced to a variety of products via different and sometimes multistep (dashed arrows represent possible multistep processes) reactions. All details are deliberately not shown. The key point is to appreciate that fermentation is a broad term not solely associated with the conversion of pyruvate to lactic acid or ethanol. Source: Marc T. Facciotti (original work)

A note on the link between substrate-level phosphorylation and fermentation 

Fermentation occurs in the absence of molecular oxygen (O₂). It is an anaerobic process. Notice there is no O₂ in any of the fermentation reactions shown above. Many of these reactions are quite ancient, hypothesized to be some of the first energy-generating metabolic reactions to evolve. This makes sense if we consider the following:

1. The early atmosphere was highly reduced, with little molecular oxygen readily available. 
2. Small, highly reduced organic molecules were relatively available, arising from a variety of chemical reactions. 
3. These types of reactions, pathways, and enzymes are found in many different types of organisms, including bacteria, archaea, and eukaryotes, suggesting these are very ancient reactions. 
4. The process evolved long before O₂ was found in the environment. 
5. The substrates, highly reduced, small organic molecules, like glucose, were readily available. 
6. The end products of many fermentation reactions are small organic acids, produced by the oxidation of the initial substrate. 
7. The process is coupled to substrate-level phosphorylation reactions. That is, small, reduced organic molecules are oxidized, and ATP is generated by first a red/ox reaction followed by the substrate-level phosphorylation. 
8. This suggests that substrate-level phosphorylation and fermentation reactions coevolved.
    

Consequences of fermentation

Imagine a world where fermentation is the primary mode for extracting energy from small molecules. As populations thrive, they reproduce and consume the abundance of small, reduced organic molecules in the environment, producing acids. One consequence is the acidification (decrease in pH) of the environment, including the internal cellular environment. This can be disruptive, since changes in pH can have a profound influence on the function and interactions among various biomolecules. Therefore, mechanisms needed to evolve that could remove the various acids. Fortunately, in an environment rich in reduced compounds, substrate-level phosphorylation and fermentation can produce large quantities of ATP. 

It is hypothesized that this scenario was the beginning of the evolution of the F₀F₁-ATPase, a molecular machine that hydrolyzes ATP and translocates protons across the membrane (we'll see this again in the next section). With the F₀F₁-ATPase, the ATP produced from fermentation could now allow for the cell to maintain pH homeostasis by coupling the free energy of hydrolysis of ATP to the transport of protons out of the cell. The downside is that cells are now pumping all of these protons into the environment, which will now start to acidify.

 

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 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 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.

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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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