Spring_2024_Bis2A_Igo_Lecture_Reading_09

Learning Objectives Associated with Spring_2024_Bis2A_Igo_Reading_09 ME.16 Apply the concept of the “conservation of mass” to metabolism by describing the different forms mass takes as it enters and leaves the cell (e.g. input: reduced molecules like glucose, lipids, proteins, etc. & output: oxidized molecules like CO2, H2O etc.).  ME.15 Apply the concept of the "conservation of energy" to central metabolism. Follow energy from "sources" of electrons with relatively low reduction potentials to "sinks" with higher redox potentials, describe the major transfers of energy and how this energy is "stored" at each stage. Learning objectives above are for the full course, but particularly relevant to the discussion of metabolism and thus extend well beyond this lecture MS.28 Identify ATP from its molecular structure and identify the core nucleotide functional units: nitrogenous base, ribose, and phosphates. MS.27 Identify the “high-energy” bonds in ATP. GC.71 Describe how the term "high energy" is commonly used in the context of molecular bonds and what is implied by its use. That is, answer": what makes a bond "high energy" and when is it appropriate to use the term? ME.3 Create a thermodynamic argument for how ATP hydrolysis can be coupled to drive endergonic reactions. ME.4 Explain the process of substrate level phosphorylation (SLP) and identify the SLP reactions when given a collection of reactions, such as in a pathway. ME.5 Explain the important contribution of water in determining the negative ΔG^0’ of the hydrolysis of a phosphoanhydride bond in ATP. ME.7 Create an "energy story" for glycolysis. The story should list the overall reactants and products, sources of energy, energy transfers, the types of reactions involved in energy transfer, and the mediators of the transformations of matter and transfers of energy. ME.11 Create an energy story explaining the functional value of pyruvate oxidation and its importance in generating building blocks for biomolecules. ME.8 Create an “energy story” explaining the functional value of pyruvate reduction (fermentation) to a cell and its importance in regenerating the NAD^+ pool. ME.14 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. ME.12 Create an energy story for each reaction in the TCA cycle. NOTE: None of the learning objectives ask you to memorize the various substrates, products and enzymes involved in Carbon Metabolism.  This information is easy to look up. Therefore, learn to explain what is going on in these pathways when you are provided with a specific set of reactions.

ATP

An important chemical compound is adenosine triphospate (ATP). The main cellular role of ATP is as a “short-term” energy transfer device for the cell. The hydrolysis reactions that liberate one or more of ATP's phosphates are exergonic and many, many cellular proteins have evolved to interact with ATP in ways that help facilitate the transfer of energy from hydrolysis to myriad other cellular functions. In this way, ATP is often called the “energy currency” of the cell: it has reasonably fixed values of energy to transfer to or from itself and can exchange that energy between many potential donors and acceptors. We will see many examples of ATP "at work" in the cell, so be looking for them. As you see them, try to think of them as functional examples of Nature's uses for ATP that you could expect to see in another reaction or context.

ATP structure and function

At the heart of ATP is the nucleotide called adenosine monophosphate (AMP). Like the other nucleotides, AMP is composed of a nitrogenous base (an adenine molecule) bonded to a ribose molecule and a single phosphate group. The addition of a second phosphate group to this core molecule results in the formation of adenosine diphosphate (ADP); the addition of a third phosphate group forms adenosine triphosphate (ATP).

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Figure 1. ATP (adenosine triphosphate) has three phosphate groups that can be removed by hydrolysis to form ADP (adenosine diphosphate) or AMP (adenosine monophosphate).

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The phosphorylation (or condensation of phosphate groups onto AMP) is an endergonic process. By contrast, the hydrolysis of one or two phosphate groups from ATP, a process called dephosphorylation, is exergonic. Why? Let's recall that the terms endergonic and exergonic refer to the sign on the difference in standard free energy of a reaction between the products and reactants, ΔG°'. Here we are explicitly assigning a direction to the reaction, either toward phosphorylation or dephosphorylation of the nucleotide. In the phosphorylation reaction, the reactants are the nucleotide and an inorganic phosphate while the products are a phosphorylated nucleotide and WATER. In the dephosphorylation/hydrolysis reaction, the reactants are the phosphorylated nucleotide and WATER while the products are inorganic phosphate and the nucleotide minus one phosphate.

Since Gibbs free energy is a state function, it doesn't matter how the reaction happens; you just consider the beginning and ending states. As an example, let's examine the hydrolysis of ATP. The reactants ATP and water are characterized by their atomic makeup and the kinds of bonds between the constituent atoms. We can associate some free energy with each of the bonds and their possible configurations—likewise for the products. If we examine the reaction from the standpoint of the products and reactants and ask "how can we recombine atoms and bonds in the reactants to get the products?," we find that a phosphoanhydride bond between an oxygen and a phosphorus must be broken in the ATP, a bond between an oxygen and hydrogen must be broken in the water, a bond must be made between the OH (that came from the splitting of water) and the phosphorus (from the freed PO₃^-2), and a bond must be formed between the H (derived from the splitting of water) and the terminal oxygen on the phosphorylated nucleotide. It is the sum of energies associated with all of those bond rearrangements (including those directly associated with water) that makes this reaction exergonic. We could make a similar analysis with the reverse reaction.

Is there something special about the specific bonds involved in these molecules? Much is made in various texts about the types of bonds between the phosphates of ATP. Certainly, the properties of the bonds in ATP help define the molecule's free energy and reactivity. However, while it is appropriate to apply concepts like charge density and availability of resonance structures to this discussion, trotting these terms out as an "explanation" without a thorough understanding of how these factors influence the free energy of the reactants is a special kind of hand-waving that we shouldn't engage in. Most BIS2A students have not had any college chemistry and those who have are not likely to have discussed those terms in any meaningful way. So, explaining the process using the ideas above only gives a false sense of understanding, assigns some mystical quality to ATP and its "special" bonds that don't exist, and distracts from the real point: the hydrolysis reaction is exergonic because of the properties of ATP and ALSO because of the chemical properties of water and those of the reaction products. For this class, it is sufficient to know that dedicated physical chemists are still studying the process of ATP hydrolysis in solution and in the context of proteins and that they are still trying to account for the key enthalpic and entropic components of the component free energies. We'll just need to accept a certain degree of mechanistic chemical ignorance and be content with a description of gross thermodynamic properties. The latter is perfectly sufficient to have deep discussions about the relevant biology.

"High-Energy" bonds

What about the term "high-energy bonds" that we so often hear associated with ATP? If there is nothing "special" about the bonds in ATP, why do we always hear the term "high-energy bonds" associated with the molecule? The answer is deceptively simple. In biology the term "high-energy bond" is used to describe an exergonic reaction involving the hydrolysis of the bond in question that results in a "large," negative change in free energy. Remember that this change in free energy has not only to do with the bond in question but rather the sum of all bond rearrangements in the reaction. What constitutes a large change? It is a rather arbitrary assignment usually associated with an amount of energy associated with the types of anabolic reactions we typically observe in biology. If there is something special about the bonds in ATP, it is not uniquely tied to the free energy of hydrolysis, as there are plenty of other bonds whose hydrolysis results in greater negative differences in free energy.

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Figure 2. The standard free energy of hydrolysis of different types of bonds can be compared to that of the hydrolysis of ATP. Source: http://bio.libretexts.org/Core/Biochemistry/Oxidation_and_Phosphorylation/ATP_and_Oxidative_Phosphorylation/Properties_of_ATP

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Table 1. Table of common cellular phosphorylated molecules and their respective standard free energies of hydrolysis.

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External link discussing the energetics of coupling ATP hydrolysis to other reactions

http://bio.libretexts.org/Core/Biochemistry/Oxidation_and_Phosphorylation/ATP_and_Oxidative_Phosphorylation/Properties_of_ATP

 

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Possible NB Discussion Point

You have just now read about two important molecules: NADH/NAD⁺ and ATP. In what biological contexts/process do you expect to see NADH/NAD⁺? What about ATP? Can you state what you know so far about the relationship between NADH/NAD⁺ and ATP? Take a moment to identify any gaps in comprehension you might have -- what questions are you left with after reading the text? Help your peers out with their questions/discussions to reinforce your own knowledge!

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The cycling of ATP pools

Estimates for the number of ATP molecules in a typical human cell range from ~3x10⁷ (~5x10^-17 moles ATP/cell) in a white blood cell to 5x10⁹ (~9x10^-15 moles ATP/cell) in an active cancer cell. While these numbers might seem large, and already amazing, consider that it is estimated that this pool of ATP turns over (becomes ADP and then back to ATP) 1.5 x per minute. Extending this analysis yields the estimate that this daily turnover amounts to roughly the equivalent of one body weight of ATP getting turned over per day. That is, if no turnover/recycling of ATP happened, it would take one body weight worth of ATP for the human body to function, hence our previous characterization of ATP as a "short-term" energy transfer device for the cell.

While the pool of ATP/ADP may be recycled, some of the energy that is transferred in the many conversions between ATP, ADP, and other biomolecules is also transferred to the environment. In order to maintain cellular energy pools, energy must transfer in from the environment as well. Where does this energy come from? The answer depends a lot on where energy is available and what mechanisms Nature has evolved to transfer energy from the environment to molecular carriers like ATP. In nearly all cases, however, the mechanism of transfer has evolved to include some form of redox chemistry.

In this and the sections that follow we are concerned with learning some critical examples of energy transfer from the environment, key types of chemistry and biological reactions involved in this process, and key biological reactions and cellular components associated with energy flow between different parts of the living system. We focus first on reactions involved in the (re)generation of ATP in the cell (not those involved in the creation of the nucleotide per se but rather those associated with the transfer of phosphates onto AMP and ADP).

Video link

For another perspective - including places you'll see ATP in Bis2a, take a look at this video (10 minutes) by clicking here.

 

How do cells generate ATP?

A variety of mechanisms have emerged over the 3.25 billion years of evolution to create ATP from ADP and AMP. The majority of these mechanisms are modifications on two themes: direct synthesis of ATP or indirect synthesis of ATP with two basic mechanisms known respectively as substrate level phosphorylation (SLP) and oxidative phosphorylation. Both mechanisms rely on biochemical reactions that transfer energy from some energy source to ADP or AMP to synthesize ATP. These topics are substantive, so they will be discussed in detail in the next few modules.

 

Glycolysis: An overview

Organisms, whether unicellular or multicellular, need to find ways of getting at least two key things from their environment: (1) matter or raw materials for maintaining a cell and building new cells and (2) energy to help with the work of staying alive and reproducing. Energy and the raw materials may come from different places. For instance, organisms that primarily harvest energy from sunlight will get raw materials for building biomolecules from sources like CO₂. By contract, some organisms rely on red/ox reactions with small molecules and/or reduced metals for energy and get their raw materials for building biomolecules from compounds unconnected to the energy source. Meanwhile, some organisms (including ourselves), have evolved to get energy AND the raw materials for building and cellular maintenance from sometimes associated sources.

Glycolysis is the first metabolic pathway discussed in BIS2A; a metabolic pathway is a series of linked biochemical reactions. Because of its ubiquity in biology, we hypothesize that glycolysis was probably one of the earliest metabolic pathways to evolve (more on this later). Glycolysis is a ten-step metabolic pathway that is centered on the processing of glucose for both energy extraction from chemical fuel and for the processing of the carbons in glucose into various other biomolecules (some of which are key precursors of many much more complicated biomolecules). We will therefore examine our study of glycolysis using the precepts outlined in the energy challenge rubric that ask us to formally consider what happens to BOTH matter and energy in this multi-step process.

The energy story and design challenge of glycolysis

Our investigation of glycolysis is a good opportunity to examine a biological process using both the energy story and the design challenge rubrics and perspectives.

The design challenge rubric will try to get you to think actively, and broadly and specifically, about why we are studying this pathway—what is so important about it? What "problems" does the evolution of a glycolytic pathway allow life to solve or overcome? We will also want to think about alternate ways to solve the same problems and why they may or may not have evolved. Later, we will examine a hypothesis for how this pathway—and other linked pathways—may have evolved, and thinking about alternative strategies for satisfying various constraints will come in handy then.

We ask you to think about glycolysis through the lens of an energy story in which you examine the 10-step process as a set of matter and energy inputs and outputs, a process with a beginning and an end. By taking this approach you will learn not only about glycolysis, but also some skills required to read and interpret other biochemical pathways.   

So what is glycolysis? Let's find out.

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Figure 1. The ten biochemical reactions of glycolysis are shown. Enzymes are labeled in blue. The structure of each sugar-derived compound is depicted as a molecular model; other reactants and products may be abbreviated (e.g., ATP, NAD+, etc.). The box surrounding the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase indicates that this reaction is of special interest in the course. Attribution: Marc T. Facciotti (original work)

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 Table 1. This table shows glycolytic enzymes and measurements of the energy at standard state (ΔG°'/(kJ/mol)) compared with measurements taken from a living cell (ΔG/(kJ/mol)). Under conditions of constant temperature and pressure, (ΔG°'/(kJ/mol)), reactions will occur in the direction that leads to a decrease in the value of the Gibbs free energy. Cellular measurements of ΔG can be dramatically different from ΔG°' measurements because of cellular conditions, such as concentrations of relevant metabolites, etc. There are three large, negative ΔG drops in the cell in the process of glycolysis. We consider these reactions irreversible and are often subject to regulation

Enzyme	Step	ΔG/(kJ/mol)	ΔG°'/(kJ/mol)
Hexokinase	1	-34	-16.7
Phosphoglucose isomerase	2	-2.9	1.67
Phosphofructokinase	3	-19	-14.2
Fructose-bisphosphate aldolase	4	-0.23	23.9
Triose phosphate isomerase	5	2.4	7.56
Glyceraldehyde 3-phosphate dehydrogenase	6	-1.29	6.30
Phosphoglycerate kinase	7	0.09	-18.9
Phosphoglycerate mutase	8	0.83	4.4
Enolase	9	1.1	1.8
Pyruvate kinase	10	-23.0	-31.7

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Overall, the glycolytic pathway comprises 10 enzyme-catalyzed steps. The primary input into this pathway is a single molecule of glucose, though we discover that other molecules may enter this pathway at various steps. We will focus our attention on (1) consequences of the overall process, (2) several key reactions that highlight important types of biochemistry and biochemical principles we will want to carry forward to other contexts, and (3) alternative fates of the intermediates and products of this pathway.

Note for reference that glycolysis is an anaerobic process. There is no requirement for molecular oxygen in glycolysis - oxygen gas is not a reactant in any of the chemical reactions in glycolysis. Glycolysis occurs in the cytosol or cytoplasm of cells. For a short (three-minute) overview YouTube video of glycolysis, click here.

First half of glycolysis: energy investment phase

We typically refer the first few steps of glycolysis as an "energy investment phase" of the pathway. This, however, doesn't make much intuitive sense (in the framework of a design challenge; it's not clear what problem this energy investment solves) if one only looks at glycolysis as an "energy-producing" pathway and until these steps of glycolysis are put into a broader metabolic context. We'll try to build that story as we go, so for now just recall that we mentioned that some first steps are often associated with energy investment and ideas like "trapping" and "commitment" that are noted in the figure below.

Step 1 of glycolysis:

The first step in glycolysis, shown below in Figure 2, is glucose being catalyzed by hexokinase, an enzyme with broad specificity that catalyzes the phosphorylation of six-carbon sugars. Hexokinase catalyzes the phosphorylation of glucose, where glucose and ATP are substrates for the reaction, producing a molecule called glucose 6-phosphate and ADP as products.

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Figure 2. The first half of glycolysis is called the energy investment phase. In this phase, the cell spends two ATPs into the reactions. Attribution: Marc T. Facciotti (original work)

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Note:
The paragraph above states that the enzyme hexokinase has "broad specificity." This means that it can catalyze reactions with different sugars, not just glucose. From a molecular perspective, can you explain why this might be the case? Does this challenge your conception of enzyme specificity? If you Google the term "enzyme promiscuity" (don't worry; it's safe for work), you will hopefully gain a broader appreciation for enzyme selectivity and activity.

The conversion of glucose to the negatively charged glucose 6-phosphate significantly reduces the likelihood that the phosphorylated glucose leaves the cell by diffusion across the hydrophobic interior of the plasma membrane. It also "marks" the glucose in a way that tags it for several possible fates (see Figure 3).

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Figure 3. Note that this figure shows that glucose 6-phosphate can, depending on cellular conditions, be directed to multiple fates. While it is a component of the glycolytic pathway, it is not only involved in glycolysis but also in the storage of energy as glycogen (colored in cyan) and in the building of various other molecules like nucleotides (colored in red). Source: Marc T. Facciotti (original work)

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As Figure 3 shows, glycolysis is but one fate for glucose 6-phosphate (G6P). Depending on cellular conditions, G6P may be diverted to the biosynthesis of glycogen (for energy storage), or it may be diverted into the pentose phosphate pathway for the biosynthesis of various biomolecules, including nucleotides. This means that G6P, while involved in the glycolytic pathway, is not solely tagged for oxidation at this phase. Perhaps showing the broader context that this molecule is involved in (in addition to the rationale that tagging glucose with a phosphate decreases the likelihood that it will leave the cell) helps to explain the seemingly contradictory (if you only consider glycolysis as an "energy-producing" process) reason for transferring energy from ATP onto glucose if it is only to be oxidized later—that is, glucose is not only used by the cell for harvesting energy and several other metabolic pathways depend on the transfer of the phosphate group.

Step 2 of glycolysis:

In the second step of glycolysis, an isomerase catalyzes the conversion of glucose 6-phosphate into one of its isomers, fructose 6-phosphate. An isomerase is an enzyme that catalyzes the conversion of a molecule into one of its isomers.

Step 3 of glycolysis:

The third step of glycolysis is the phosphorylation of fructose 6-phosphate, catalyzed by the enzyme phosphofructokinase. A second ATP molecule donates a phosphate to fructose 6-phosphate, producing fructose 1,6-bisphosphate and ADP as products. In this pathway, phosphofructokinase is a rate-limiting enzyme, and its activity is tightly regulated. It is allosterically activated by AMP when the concentration of AMP is high and when it is moderately allosterically inhibited by ATP at the same site. Citrate, a compound we'll discuss soon, also acts as a negative allosteric regulator of this enzyme. In this way, phosphofructokinase monitors or senses molecular indicators of the energy status of the cells and can in response act as a switch that turns on or off the flow of the substrate through the rest of the metabolic pathway depending on whether there is “sufficient” ATP in the system. The conversion of fructose 6-phosphate into fructose 1,6-bisphosphate is sometimes referred to as a commitment step by the cell to the oxidation of the molecule in the rest of the glycolytic pathway by creating a substrate for and helping to energetically drive the next highly endergonic (under standard conditions) step of the pathway.

Step 4 of glycolysis:

In the fourth step in glycolysis, an enzyme, fructose-bisphosphate aldolase, cleaves 1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

Second half: energy payoff phase

If viewed in the absence of other metabolic pathways, glycolysis has so far cost the cell two ATP molecules and produced two small, three-carbon sugar molecules: dihydroxyacetone phosphate (DAP) and glyceraldehyde 3-phosphate (G3P). When viewed in a broader context, this investment of energy to produce a variety of molecules that can be used in a variety of other pathways doesn't seem like such a bad investment.

Both DAP and G3P can proceed through the second half of glycolysis. We now examine these reactions.

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Figure 4. The second half of glycolysis is called the energy payoff phase. In this phase, the cell gains two ATP and two NADH compounds. At the end of this phase, glucose has become partially oxidized to form pyruvate. Attribution: Marc T. Facciotti (original work).

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Step 5 of glycolysis:

In the fifth step of glycolysis, an isomerase transforms the dihydroxyacetone phosphate into its isomer, glyceraldehyde 3-phosphate. The six-carbon glucose has therefore now been converted into two phosphorylated three-carbon molecules of G3P.

Step 6 of glycolysis:

The sixth step is key and one from which we can now leverage our understanding of the several chemical reactions that we've studied so far. If you're energy focused, this is finally a step of glycolysis where some reduced sugar becomes oxidized. The reaction is catalyzed by the enzyme glyceraldehyde 3-phosphate dehydrogenase. This enzyme catalyzes a multi-step reaction between three substrates—glyceraldehyde 3-phosphate, the cofactor NAD⁺, and inorganic phosphate (P_i)—and produces three products: 1,3-bisphosphoglycerate, NADH, and H⁺. One can think of this reaction as two reactions: (1) an oxidation/reduction reaction and (2) a condensation reaction in which an inorganic phosphate is transferred onto a molecule. Here, the red/ox reaction, a transfer of electrons off G3P and onto NAD⁺, is exergonic, and the phosphate transfer is endergonic. The net standard free energy change hovers around zero—more on this later. The enzyme here acts as a molecular coupling agent to couple the energetics of the exergonic reaction to that of the endergonic reaction, thus driving both forward. This processes happens through a multi-step mechanism in the enzyme's active site and involves the chemical activity of a variety of functional groups.

It is important to note that this reaction depends upon the availability of the oxidized form of the electron carrier, NAD⁺. If we consider that there is a limiting pool of NAD⁺, we can then conclude that the reduced form of the carrier (NADH) must continuously oxidize back into NAD⁺ to keep this step going. If NAD⁺ is not available, the second half of glycolysis slows down or stops.

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Possible NB Discussion Point

Can you write an energy story for Step 6 of glycolysis (the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase)? When you discuss energy, just describe whether steps are exergonic or endergonic. As a group, try to build up an increasingly "expert" version that is complete, brief, and uses appropriate vocabulary.  Amend one another's texts in a polite and constructive way.

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Step 7 of glycolysis:

In the seventh step of glycolysis, catalyzed by phosphoglycerate kinase (an enzyme named for the reverse reaction), 1,3-bisphosphoglycerate transfers a phosphate to ADP, forming one molecule of ATP and a molecule of 3-phosphoglycerate. This reaction is exergonic and is also an example of substrate-level phosphorylation.

Step 8 of glycolysis:

In the eighth step, the remaining phosphate group in 3-phosphoglycerate moves from the third carbon to the second carbon, producing 2-phosphoglycerate (an isomer of 3-phosphoglycerate). The enzyme catalyzing this step is a mutase (isomerase).

Step 9 of glycolysis:

Enolase catalyzes the ninth step. This enzyme causes 2-phosphoglycerate to lose water from its structure; this is a dehydration reaction, resulting in the formation of a double bond that increases the potential energy in the remaining phosphate bond and produces phosphoenolpyruvate (PEP).

Step 10 of glycolysis:

The last step in glycolysis is catalyzed by the enzyme pyruvate kinase (the enzyme in this case is named for the reverse reaction of pyruvate’s conversion into PEP) and results in the production of a second ATP molecule by substrate-level phosphorylation and the compound pyruvic acid (or its salt form, pyruvate). Many enzymes in enzymatic pathways are named for the reverse reactions, since the enzyme can catalyze both forward and reverse reactions (these may have been described initially by the reverse reaction that takes place in vitro, under non-physiological conditions).

Outcomes of glycolysis

Here are a couple of things to consider:

One of the clear outcomes of glycolysis is the biosynthesis of compounds that can enter into a variety of metabolic pathways. Likewise, compounds coming from other metabolic pathways can feed into glycolysis at various points. So, this pathway can be part of a central exchange for carbon flux within the cell.

If glycolysis runs long enough, the constant oxidation of glucose with NAD⁺ can leave the cell with a problem: how to regenerate NAD⁺ from the two molecules of NADH produced. If the cell does not regenerate  NAD⁺, nearly all the cell's NAD+ will transform into NADH. So how do cells regenerate NAD⁺?

Pyruvate is not completely oxidized; There is still some energy to extract. How might this happen? Also, what should the cell do with all of that NADH? Is there any energy there to extract?

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Possible NB Discussion Point

To some, that glycolysis is such a complex, multi-step pathway may seem counter-intuitive: “Why wouldn’t evolution lead to a *simpler* way to extract energy from food since energy is an important requirement for life?”  Explain the necessity/advantage of having glucose get broken down in many steps.

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Substrate-level phosphorylation (SLP)

The simplest route to synthesize ATP is substrate-level phosphorylation. ATP molecules are generated (that is, regenerated from ADP) because of a chemical reaction that occurs in catabolic pathways. A phosphate group is removed from an intermediate reactant in the pathway, and the free energy of the reaction is used to add the third phosphate to an available ADP molecule, producing ATP. This very direct method of phosphorylation is called substrate-level phosphorylation (SLP). We can find SLP in a variety of catabolic reactions, most notably in two specific reactions in glycolysis (which we will discuss specifically later). What the reaction requires is a high-energy intermediate compound whose free energy of oxidation can drive the synthesis of ATP.

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Figure 5. Here is one example of substrate-level phosphorylation occurring in glycolysis. There is a direct transfer of a phosphate group from the carbon compound onto ADP to form ATP. Attribution: Marc T. Facciotti (own work)

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In this reaction, the reactants are a phosphorylated carbon compound called G3P (from step 6 of glycolysis) and an ADP molecule, and the products are 1,3-BPG and ATP. The transfer of the phosphate from G3P to ADP to form ATP in the active site of the enzyme is substrate-level phosphorylation. This occurs twice in glycolysis and once in the TCA cycle (for a subsequent reading).

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:

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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)

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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:

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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)

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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.

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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)

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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.

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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.

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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.

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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)

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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 complete TCA cycle.

Since all cells require the ability of make these precursor molecules, one might expect that all organisms would have a fully complete 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

 

PRACTICE POST GUIDE

General Practice

3. Why: I am repeating this exercise from the previous lecture - it’s that important. Redox is central to metabolism and energy flow. This topic also has some of the seemingly easy but, nevertheless, most confusing vocabulary.  It is a good idea to learn the vocabulary as quickly as possible and to associate them with any basic redox reaction.

How to practice: I can’t stress enough - again - how important it will be for you to learn the vocabulary associated with redox chemistry (oxidized, reduced, oxidizing agent, reducing agent etc.). We will be using these terms frequently during this part of the course. Class, discussion, and the reading will be much more confusing if you do not have a good understanding of the terms. Remember, we don’t need to do any “fancy” chemistry and find the actual oxidation states of biomolecules—we just need to be able to recognize that there is a flow of electrons (a loss of an electron(s) from one compound and a corresponding gain of an electron(s) by another compound) between compounds.   

This exercise helps you practice learning objectives: GC.29 Given a redox reaction, identify the reducing agent, oxidizing agent, molecule that becomes oxidized, and the reduced species. Identify which species the electron(s) "starts" in, and to which species it "goes."

 

4. Why: ATP is a very important molecule for the short-term transfer of energy, energy coupling, and transfer of phosphate functional groups.  It’s also a core building block of RNA and in a deoxy-form core building block of RNA.  It’s an important molecule to get to know better.  Here we start by asking you to use the global textbook - the internet - to find reactions involving ATP.  Google or your favorite search engine is good for that.  This website <http://biochemical-pathways.com/#/map/1> allows you to browse biochemical maps - use that to find reactions involving ATP.

How to practice: Look up some metabolic pathways on the internet, in your textbook, and/or on the metabolic chart located in the learning center. See how many different reactions involve ATP/ADP? What can you say about these coupled reactions? Can you dissect the reactions you find on the metabolic charts and tell their energy stories?  Do this for a few.

This exercise helps you practice learning objectives: ME.3 Create a thermodynamic argument for how ATP hydrolysis can be coupled to drive endergonic reactions.; ME.4 Explain the process of substrate level phosphorylation (SLP) and identify the SLP reactions when given a collection of reactions, such as in a pathway.

 

5. Why: More practice examining ATP and its role in energy transfer. Don’t need to say more.

How to practice: The diagram below shows a number of important features about ATP/ADP. Rewrite this diagram for yourself - in your notes.  Take special care to deconstruct ATP into its three core parts and to identify the so-called “high-energy” bonds. Now think about the hydrolysis reaction depicted specifically.  The water is not drawn explicitly in the figure - try to find where it shows up.  You’ll do more in part c.

1. 1. The reaction ATP + 2H₂O —> ADP+Pi + H₃O⁺ is exergonic. What are some of the other important features of this reaction?
   2. The reaction ADP+Pi + H₃O⁺ —> ATP + 2 H₂O is endergonic. What are some of the other important features of this reaction?
   3. Notice that both H₂O and H₃O⁺ are not shown in the drawing. Can you draw them in?

This exercise helps you practice learning objectives: MS.28 Identify ATP from its molecular structure and identify the core nucleotide functional units: nitrogenous base, ribose, and phosphates.; MS.27 Identify the “high-energy” bonds in ATP.; ME.3 Create a thermodynamic argument for how ATP hydrolysis can be coupled to drive endergonic reactions.; ME.5 Explain the important contribution of water in determining the negative ΔG⁰’ of the hydrolysis of a phosphoanhydride bond in ATP.; GC.71 Describe how the term "high energy" is commonly used in the context of molecular bonds and what is implied by its use. That is, answer": what makes a bond "high energy" and when is it appropriate to use the term?

 

 

 

6. Why: This is extending the practice from exercise 5 but now linking back to previous skills involving reaction coordinate diagrams and thermodynamics.

How to practice: Draw the reaction coordinate diagrams for the following reactions:

1. 1. ATP + 2H₂O —> ADP+Pi + H₃O⁺. Explain the key features of this reaction and why it is important to cells.
   2. b. ADP+Pi + H₃O⁺ —> ATP + 2H₂O. Explain the key features of this reaction and why this reaction is important to cells.

This exercise helps you practice learning objectives: ME.5 Explain the important contribution of water in determining the negative ΔG⁰’ of the hydrolysis of a phosphoanhydride bond in ATP.

 

7. Why: The direct transfer of phosphate groups from small molecules to ADP to make ATP is known as substrate-level phosphorylation. This is one of the main mechanisms for ATP synthesis that you need to learn how to spot.

How to practice: Examine the glycolytic pathway. Identify all instances of substrate-level phosphorylation and create an energy story for those reactions.

This exercise helps you practice learning objectives: ME.4 Explain the process of substrate level phosphorylation (SLP) and identify the SLP reactions when given a collection of reactions, such as in a pathway.

 

8. Why: Glycolysis is a universal metabolic pathway of carbon flow and energy transfer.  Here we want to start to familiarize ourselves with this set of reactions and some of its core properties.  This question ask you to think about different parts of the pathway and how they connect.

How to practice: In steps 6 through 10 of glycolysis, the conversion of one mole of glyceraldehyde-3-phosphate to pyruvate yields two moles of ATP. However, the oxidation of glucose to pyruvate produces a total of four moles of ATP. Where do the remaining two moles of ATP come from?

This exercise helps you practice learning objectives: ME.7 Create an "energy story" for glycolysis. The story should list the overall reactants and products, sources of energy, energy transfers, the types of reactions involved in energy transfer, and the mediators of the transformations of matter and transfers of energy.; ME.16 Apply the concept of the “conservation of mass” to metabolism by describing the different forms mass takes as it enters and leaves the cell (e.g. input: reduced molecules like glucose, lipids, proteins, etc. & output: oxidized molecules like CO₂, H₂O etc.). 

 

9. Why: One of the “big” lessons in Bis2a learning how to connect different lessons and concepts together. Nowhere is it more straightforward to make connections - and to think about what it means for the “bigger picture” - that in our discussion of central metabolism. To really “get it”, however, you need to start building a mental picture of the connections between the core pathways we talk about in the class.  Practice that here.

How to practice: I suggest that you practice recreating a high-level overview of glycolysis and TCA cycle. Let’s do that here!

1. 1. Try to write down the two high-level questions that we wanted to ask with our discussion of glycolysis. From a Design Challenge perspective, what were the two key problems that we wanted to think more about?
   2. We talked about one of these problems at length in the context of a redox reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. Here, try to summarize that reactions’ energy story and recreate it from scratch if you can. Use our new vocabulary when you think it helps your explanation. The story includes concepts from redox chemistry, energetics, and new bond types, as well as comparisons of free-energies of hydrolysis.
   3. The second part of the story of glycolysis has to do with some of the special 12 compounds that make “everything”—summarize it.

This exercise helps you practice learning objectives: ME.16 Apply the concept of the “conservation of mass” to metabolism by describing the different forms mass takes as it enters and leaves the cell (e.g. input: reduced molecules like glucose, lipids, proteins, etc. & output: oxidized molecules like CO₂, H₂O etc.).; ME.15 Apply the concept of the "conservation of energy" to central metabolism. Follow energy from "sources" of electrons with relatively low reduction potentials to "sinks" with higher redox potentials, describe the major transfers of energy and how this energy is "stored" at each stage.; ME.14 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.; ME.10 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.; ME.7 Create an "energy story" for glycolysis. The story should list the overall reactants and products, sources of energy, energy transfers, the types of reactions involved in energy transfer, and the mediators of the transformations of matter and transfers of energy.

 

10. Why: ATP is a crucially important molecule in biological systems - it plays many different roles. Here we want to review its role as an energy carrier and make sure we understand some of the basis of how “it works”.

How to practice: As discussed in the “book”, we often talk about the hydrolysis of ATP to ADP+Pi as an exergonic process. Your chemistry professor will correctly tell you that breaking bonds takes energy. How can both of these observations be true? What is the distinction between saying that we “hydrolyze ATP" and “breaking the phosphate bond” that makes both of the previous statements true? Why does the teaching staff take great pains to note the the “hydrolysis of certain bonds” is energetically favorable?

This exercise helps you practice learning objectives: ME.5 Explain the important contribution of water in determining the negative ΔG⁰’ of the hydrolysis of a phosphoanhydride bond in ATP.; MS.27 Identify the “high-energy” bonds in ATP. - These learning goals carry over from the previous lecture, but we’ll keep practicing here!

 

11. Why: Fermentation is a key process for recycling NADH into NAD⁺ and in the production of many

How to practice: In the reading assignments and in lecture, you were given the problem of figuring out what options the cell might reasonably have to re-oxidize the NADH to NAD⁺ in order to avoid consuming the available pools of NAD⁺ and thus stopping glycolysis. In today’s lecture we discussed one solution—fermentation.

1. What is your definition of fermentation? Does it relate to oxygen or to regenerating NAD⁺ pools?
2. Can fermentation happen in the presence of oxygen?.
3. Is fermentation the same thing as anaerobic respiration?
4. We will revisit this topic again so start thinking about it. Be aware that the last two parts of this question refer to a very common misconception.

This exercise helps you practice learning objectives:  ME.8 Create an “energy story” explaining the functional value of pyruvate reduction (fermentation) to a cell and its importance in regenerating the NAD⁺ pool.