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SS1_2018_Lecture_05

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    Metabolism in BIS2A

    Cellular metabolism represents roughly 1/3 of the BIS2A curriculum. While this may seem like a lot, we cover very little of what a classic course in metabolism (like BIS103) will cover, and an even smaller (really minuscule) fraction of the metabolism that occurs on the planet. What we do cover, however, is very important foundational knowledge. You will learn about some common chemical reactions that are associated with the transformation of life's molecular building blocks and about different core modes of energy transfer that you will encounter often in biology. The energy story and the design challenge rubrics introduced earlier will become increasingly important in these next few modules and beyond.

    What have we learned? How will it relate to metabolism?

    1. We have focused on the identification and chemical properties of common biological functional groups. As we dive into metabolism, this will help you be familiar with and sometimes even predict the chemical nature/reactivity of compounds you have never seen before.
    2. We have practiced recognizing and classifying molecules into four major functional groups. This will help you as we begin to discuss how to build and break down these molecules.
    3. We have learned some basic thermodynamics, giving us a common set of concepts with which to discuss whether a biochemical reaction or process is likely to occur, and if so in which direction and how fast. This will be critical as we begin to consider some of the key types of reactions that take place in metabolism.
    4. We have learned and practiced the energy story rubric. This too will allow us to systematically examine new biochemical reactions and processes and to discuss them with a common language and approach that is consistent and reinforces the lessons we learned about thermodynamics.

    A brief overview of this section

    • You will be introduced to an important concept called reduction potential and you will be given the opportunity to use a redox tower. There is also a discussion on redox chemistry in your discussion manual. Make sure to use both resources.
    • You will be introduced to two major players in metabolism, ATP and NADH. You will be expected to recognize their structures if shown on an exam.
    • The metabolic pathway glycolysis will be covered in detail. Keep in mind that we want you to be able to look at any reaction and tell us an energy story of that reaction. By no means should you spend time trying to memorize these pathways (though it will help tremendously to remember some big picture things - these will be stressed). Often we will give you the pathway as a figure on the exams. Glycolysis ultimately produces 2 ATP via a process called substrate level phosphorylation, 2 NADH and 2 pyruvate compounds.
    • We will use the reactions of the TCA cycle to create multiple examples of energy stories. The TCA cycle will also produce more ATP, NADH and completely oxidize glucose into CO2.
    • We will look at an alternative pathway to that of the TCA cycle, fermentation. In fermentation for the first time we will see NADH used as a reactant in a metabolic reaction.
    • We will follow NADH to the end of its journey, as it donates its electrons to the electron transport chain (ETC). In this module you will need to be able to use a redox tower. The ETC produces a proton gradient. No ATP is directly generated in this process. However, the proton gradient is then used by the cell (among other things) to run an enzyme called ATP synthase which catalyzes the reaction ADP + Pi --> ATP. This method of ATP production (called oxidative respiration) results in much more ATP being produced than substrate level phosphorylation.
    • And finally, we will go through the process of photosynthesis.

    Reduction-Oxidation Reactions

    In this class, the majority of the oxidation/reduction reactions reactions that we discuss occur in the context of metabolic pathways (connected sets of metabolic reactions) where compounds may be consumed by the cell, broken down into smaller parts and then reassembled into larger macromolecules.

    Lets start with some generic reactions

    Transferring electrons between two compounds results in one of these compounds loosing an electron, and one of the compounds gaining an electron. For example, look at the figure below. If we use the energy story rubric to look at the overall reaction we can compare the before and after characteristics of the reactants and products. What happens to the matter (stuff) before and after the reaction? Compound A starts as neutral and becomes positively charged. Compound B starts as neutral and becomes negatively charged. Because electrons are negatively charged, we can follow the movement of electrons from compound A to B by looking at the change in charge. A looses an electron (becoming positively charged), and in so doing we say that A has become oxidized. Oxidation is associated with the loss of electron(s). B gains the electron (becoming negatively charged), and we say that B has become reduced. Reduction is associated with gain of electrons. We also know, since something happened that energy must have been either transferred and/or reorganized in this process and we'll consider this shortly.

    generic.jpg

    Figure 1. A generic red/ox reaction. The full reaction is A +B goes to A+ + B-. The two half reactions are shown in the blue box. A is oxidized by the reaction and B is reduced by the reaction.

    Put another way, when an electron(s) is lost, or a molecule is oxidized, the electron(s) must then passed to another molecule. The molecule gaining the electron is said to be reduced. ***The oxidation and reduction reactions are always paired in what is known as an oxidation-reduction reaction (also called a red/ox reaction).****

    In Bis2A we expect you to become familiar with this terminology. Try to learn it and learn to use it as soon as possible - we will use the terms frequently and will not have the time to define them each time.

    Remember the Definitions:

    Screen Shot 2017-01-08 at 7.04.42 PM.png

    The Half Reaction

    To formalize our common understanding of red/ox reactions, we introduce the concept of the half reaction. Two half reactions are required to make the full red/ox reaction. Each half reaction can be thought of as a description of what happens to one of the two molecules involved in the red/ox reaction. This is illustrated below. In this example compound AH is being oxidized by compound B+; electrons are moving from AH to B+ to generate A+ and BH. Each reaction can be thought of as two half reactions: Where AH is being oxidized and a second reaction where B+ is being reduced to BH. These two reactions are considered coupled, a term that indicates that these two reactions occur together, at the same time.

    figure1.jpg

    Figure 2. Generic red/ox reaction where compound AH is being oxidized by compound B+. Each half reaction represents a single species or compound to either lose or gain electrons (and a subsequent proton as shown in the figure above). In half reaction #1 AH loses a proton and 2 electrons: in the second half reaction, B+ gains 2 electrons and a proton. In this example HA is oxidized to A+ while B+ is reduced to BH.

    Possible discussion

    If you consider a generic red/ox reaction and reflect back on the thermodynamic lectures what factor will determine whether a red/ox reaction will "go" in a particular direction spontaneously and what might determine its rate?

    Reduction Potential

    By convention we analyze and describe red/ox reactions with respect to reduction potentials, a term that quantitatively describes the "ability" of a compound to gain electrons. This value of the reduction potential is determined experimentally but for the purpose of this course we assume that the reader will accept that the reported values are reasonably correct. We can anthropomorphize the reduction potential by saying that it is related to the strength with which a compound can “attract” or “pull” or “capture” electrons. Not surprisingly this is is related to but not identical to electronegativity.

    What is this intrinsic property to attract electrons?

    Different compounds, based on their structure and atomic composition have intrinsic and distinct attractions for electrons. This quality is termed reduction potential or E0’and is a relative quantity (relative by comparison to some “standard” reaction). If a test compound has a stronger "attraction" to electrons than the standard (if the two competed the test compound would "take" electrons from the standard compound), we say that the test compound has a positive reduction potential whose magnitude is proportional to how much more it "wants" electrons than the standard compound. The relative strength of the compound in comparison to the standard is measured and reported in units of Volts (V)(sometimes written as electron volts or eV) or milliVolts (mV). The reference compound in most red/ox towers is H2.

    Possible discussion

    Rephrase for yourself: How do you describe or think about the difference between the concept of electronegativity and red/ox potential?

    The Red/ox Tower

    All kinds of compounds can participate in red/ox reactions. A tool has been developed to graphically tabulate red/ox half reactions based on their E0' values and to help us predict the direction of electron flow between potential electron donors and acceptors. Whether a particular compound can act as an electron donor (reductant) or electron acceptor (oxidant) depends critically on what other compound it is interacting with. The electron tower usually ranks a variety of common compounds (their half reactions) from most negative E0', compounds that readily get rid of electrons, to the most positive E0', compounds most likely to accept electrons. In addition, each half reaction is written by convention with the oxidized form on the left/followed by the reduced form on the right of the slash.
    For example the half reaction for the reduction of NAD+ to NADH is written:
    NAD+/NADH. In the tower below, the number of electrons that are transferred is also listed. For example the reduction of NAD+ to NADH involves two electrons, written in the table as 2e-.

    An biochemical electron tower is shown below.

    oxidized form

    reduced form

    n (electrons)

    Eo´ (volts)

    PS1* (ox)

    PS1* (red)

    -

    -1.20

    Acetate + CO2

    pyruvate

    2

    -0.7

    ferredoxin (ox) version 1

    ferredoxin (red) version 1

    1

    -0.7

    succinate + CO2 + 2H+

    a-ketoglutarate + H2O

    2

    -0.67

    PSII* (ox)

    PSII* (red)

    -

    -0.67

    P840* (ox)

    PS840* (red)

    -

    -0.67

    acetate

    acetaldehyde

    2

    -0.6

    glycerate-3-P

    glyceraldehyde-3-P + H2O

    2

    -0.55

    O2

    O2-

    1

    -0.45

    ferredoxin (ox) version 2

    ferredoxin (red) version 2

    1

    -0.43

    CO2

    glucose

    24

    -0.43

    CO2

    formate

    2

    -0.42

    2H+

    H2

    2

    -0.42 (at [H+] = 10-7; pH=7)

    α-ketoglutarate + CO2 + 2H+

    isocitrate

    2

    -0.38

    acetoacetate

    b-hydroxybutyrate

    2

    -0.35

    Cystine

    cysteine

    2

    -0.34

    Pyruvate + CO2

    malate

    2

    -0.33

    NAD+ + 2H+

    NADH + H+

    2

    -0.32

    NADP+ + 2H+

    NADPH + H+

    2

    -0.32

    Complex I FMN (enzyme bound)

    FMNH2

    2

    -0.3

    Lipoic acid, (ox)

    Lipoic acid, (red)

    2

    -0.29

    1,3 bisphosphoglycerate + 2H+

    glyceraldehyde-3-P + Pi

    2

    -0.29

    Glutathione, (ox)

    Glutathione, (red)

    2

    -0.23

    FAD+ (free) + 2H+

    FADH2

    2

    -0.22

    Acetaldehyde + 2H+

    ethanol

    2

    -0.2

    Pyruvate + 2H+

    lactate

    2

    -0.19

    Oxalacetate + 2H+

    malate

    2

    -0.17

    α-ketoglutarate + NH4+

    glutamate

    2

    -0.14

    FAD+ + 2H+ (bound)

    FADH2 (bound)

    2

    0.003-0.09

    Methylene blue, (ox)

    Methylene blue, (red)

    2

    0.01

    Fumarate + 2H+

    succinate

    2

    0.03

    CoQ (Ubiquinone - UQ + H+)

    UQH.

    1

    0.031

    UQ + 2H+

    UQH2

    2

    0.06

    Dehydroascorbic acid

    ascorbic acid

    2

    0.06

    Plastoquinone; (ox)

    Plastoquinone; (red)

    -

    0.08

    Ubiquinone; (ox)

    Ubiquinone; (red)

    2

    0.1

    Complex III Cytochrome b2; Fe3+

    Cytochrome b2; Fe2+

    1

    0.12

    Fe3+ (pH = 7)

    Fe2+ (pH = 7)

    1

    0.20

    Complex III Cytochrome c1; Fe3+

    Cytochrome c1; Fe2+

    1

    0.22

    Cytochrome c; Fe3+

    Cytochrome c; Fe2+

    1

    0.25

    Complex IV Cytochrome a; Fe3+

    Cytochrome a; Fe2+

    1

    0.29

    1/2 O2 + H2O

    H2O2

    2

    0.3

    P840GS (ox)

    PS840GS (red)

    -

    0.33

    Complex IV Cytochrome a3; Fe3+

    Cytochrome a3; Fe2+

    1

    0.35

    Ferricyanide

    ferrocyanide

    2

    0.36

    Cytochrome f; Fe3+

    Cytochrome f; Fe2+

    1

    0.37

    PSIGS (ox)

    PSIGS (red)

    .

    0.37

    Nitrate

    nitrite

    1

    0.42

    Fe3+ (pH = 2)

    Fe2+ (pH = 2)

    1

    0.77

    1/2 O2 + 2H+

    H2O

    2

    0.816

    PSIIGS (ox)

    PSIIGS (red)

    -

    1.10

    * Excited State, after absorbing a photon of light

    GS Ground State, state prior to absorbing a photon of light

    PS1: Oxygenic photosystem I

    P840: Bacterial reaction center containing bacteriochlorophyll (anoxygenic)

    PSII: Oxygenic photosystem II

    Table 1. Common Red/ox tower used in Bis2A. By convention the tower half reactions are written with the oxidized form of the compound on the left and the reduced form on the right. Compounds that make good electron donors have highly negative reduction potentials. Compounds such as Glucose and Hydrogen gas are excellent electron donors. By contrast compounds that make excellent electron acceptors, such as Oxygen and Nitrite have .

    Video on electron tower

    For a short video on how to use the electron tower in red/ox problems click here or below. This video was made by Dr. Easlon for Bis2A students. (This is quite informative.)

    redox_splash_screen.png

    What is the relationship between ΔE0' and ΔG?

    The question now becomes: how do we know if any given red/ox reaction is energetically spontaneous or not (exergonic or endergonic) and regardless of direction, what the free energy difference is? The answer lies in the difference in the reduction potentials of the two compounds. The difference in the reduction potential for the reaction or E0' for the reaction, is the difference between the E0' for the oxidant (the compound getting the electrons and causing the oxidation of the other compound) and the reductant (the compound losing the electrons). In our generic example below, AH is the reductant and B+ is the oxidant. Electrons are moving from AH to B+. Using the E0' of -0.32 for the reductant and +0.82 for the oxidant the total change in E0' or ΔE0' is 1.14 eV.

    figure11.jpg

    Figure 3. Generic red/ox reaction with half reactions written with reduction potential (E0') of the two half reactions indicated.

    The change in ΔE0' correlates to changes in Gibbs free energy, ΔG. In general a large positive ΔE0' is proportional to a large negative ΔG. The reactions are exergonic and spontaneous. For a reaction to be exergonic the reaction needs to have a negative change in free energy or -ΔG, this will correspond to a positive ΔE0'. In other words, when electrons flow "downhill" in a red/ox reaction from a compound with a lower (more negative) reduction potential to a second compound with a larger (more positive) reduction potential, they release free energy. The greater the voltage, E0', between the two components, the greater the energy available when electron flow occurs. It is, in fact, possible to quantify the amount of free energy available. The relationship is given by the Nernst equation:

    nernst.png

    Figure 4. The Nernst equation relates free energy of a red/ox reaction to the difference in reduction potential between the reduced products of the reaction and oxidized reactant.
    Attribution: Marc T. Facciotti

    Where:

    • n is the number of moles of electrons transferred
    • F is the Faraday constant of 96.485 kJ/V. Sometimes it is given in units of kcal/V which is 23.062 kcal/V, which is the amount of energy (in kJ or kcal) released when one mole of electrons passes through a potential drop of 1 volt

    Note

    What you should notice is that ΔG and ΔE have an inverse relationship: When ΔG is positive, ΔE is negative and when ΔG is negative, ΔE is positive. For additional review see the red/ox discussion in the Bis2A Discussion Manual.

    Introduction to Mobile Energy Carriers

    Section Summary

    Energy is moved around and transferred within the cell in a variety of ways. One critical mechanism that nature has developed is the use of recyclable molecular energy carriers. While there are several major recyclable energy carriers, they all share some common functional features:

    Properties of Key Cellular Molecular Energy Carriers

    • We think of the energy carriers as existing in "pools" of available carriers. One could, by analogy, consider these mobile energy carriers analogous to the delivery vehicles of parcel carriersthe company has a certain "pool" of available vehicles at any one time to pickup and make deliveries.
    • Each individual carrier in the pool can exist in one of multiple distinct states: it is either carrying a "load" of energy, a fractional load, or is "empty". The molecule can interconvert between "loaded" and empty and thus can be recycled. Again by analogy, the delivery vehicles can be either carrying packages or be empty and switch between these states.
    • The balance or ratio in the pool between "loaded" and "unloaded" carriers is important for cellular function, is regulated by the cell, and can often tell us something about the state of a cell. Likewise, a parcel carrier service keeps close tabs on how full or empty their delivery vehicles areif they are too full, there may be insufficient "empty" trucks to pick up new packages; if they are too empty, business must not be going well or it is shut down. There is an appropriate balance for different situations.

    In this course, we will examine two major types of molecular recyclable energy carriers: (1) the adenine nucleotides, specifically: nicotinamide adenine dinucleotide (NAD+), a close relative, nicotinamide adenine dinucleotide phosphate (NADP+), and flavin adenine dinucleotide (FAD2+) and (2) nucleotide mono-, di-, and triphosphates, with particular attention paid to adenosine triphosphate (ATP). Each of these two types of molecules is involved in energy transfer that involves different classes of chemical reactions. Adenine nucleotides are primarily associated with redox chemistry, while the nucleotide triphosphates are associated with transfers of energy that are linked to the hydrolysis or condensation of inorganic phosphates.

    Red/ox chemistry and electron carriers

    The oxidation of, or removal of an electron from, a molecule (whether accompanied with the removal of an accompanying proton or not) results in a change of free energy for that molecule—matter, internal energy, and entropy have all changed in the process. Likewise, the reduction of (the gain of electron on) a molecule also changes its free energy. The magnitude of change in free energy and its direction (positive or negative) for a red/ox reaction dictates the spontaneity of the reaction and how much energy is transferred. In biological systems, where a great deal of energy transfer happens via red/ox reactions, it is important to understand how these reactions are mediated and to begin to start considering ideas or hypotheses for why these reactions are mediated in many cases by a small family of electron carriers.

    Note: possible discussion

    Relate the burning of (the full oxidation of the sugar in) a gummy bear with the last paragraph above. What does that demonstration have to do with our upcoming discussion on red/ox carriers? There is some mention above already—can you find it?

    splash_gummy_bear.png

    Note: possible discussion

    The problem alluded to in the previous discussion question is a great place to start bringing in the design challenge rubric. If you recall, the first step of the rubric asks that you define a problem or question. In this case, let's imagine that there is a problem to define for which the mobile electron carriers below helped Nature solve.

    ***Remember, evolution DOES NOT forward-engineer solutions to problems, but in retrospect, we can use our imagination and logic to infer that what we see preserved by natural selection provided a selective advantage, because the natural innovation "solved" a problem that limited success.***

    Design challenge for red/ox carriers

    • What was a problem(s) that the evolution of mobile electron/red/ox carriers helped solve?
    • The next step of the design challenge asks you to identify criteria for successful solutions. What are criteria for success in the problem you've identified?
    • Step 3 in the design challenge asks you to identify possible solutions. Well, here Nature has identified some for us—we consider three in the reading below. It looks like Nature is happy to have multiple solutions to the problem.
    • The penultimate step of the design challenge rubric asks you to evaluate the proposed solutions against the criteria for success. This should make you think/discuss about why there are multiple different electron carriers. Are there different criteria for success? Are they each solving slightly different problems? What do you think? Be on the lookout as we go through metabolism for clues.

    NAD+/H and FADH/H2

    In living systems, a small class of compounds function as electron shuttles: they bind and carry electrons between compounds in different metabolic pathways. The principal electron carriers we will consider are derived from the B vitamin group and are derivatives of nucleotides. These compounds can be both reduced (that is, they accept electrons) or oxidized (they lose electrons) depending on the reduction potential of a potential electron donor or acceptor that they might transfer electrons to and from. Nicotinamide adenine dinucleotide (NAD+) (the structure is shown below) is derived from vitamin B3, niacin. NAD+ is the oxidized form of the molecule; NADH is the reduced form of the molecule after it has accepted two electrons and a proton (which together are the equivalent of a hydrogen atom with an extra electron).

    We are expecting you to memorize the two forms of NAD+/NADH, know which form is oxidized and which is reduced, and be able to recognize either form on the spot in the context of a chemical reaction.

    NAD+ can accept electrons from an organic molecule according to the general equation:

    redox2.png

    Here is some vocabulary review: when electrons are added to a compound, the compound is said to have been reduced. A compound that reduces (donates electrons to) another is called a reducing agent. In the above equation, RH is a reducing agent, and NAD+ is reduced to NADH. When electrons are removed from a compound, it becomes oxidized. A compound that oxidizes another is called an oxidizing agent. In the above equation, NAD+ is an oxidizing agent, and RH is oxidized to R. Put another way, the reducing agent gets oxidized and the oxidizing agent gets reduced.

    You need to get this down! We will (a) test specifically on your ability to do so (as "easy" questions), and (b) we will use the terms with the expectation that you know what they mean and can relate them to biochemical reactions correctly (in class and on tests).

    You will also encounter a second variation of NAD+, NADP+. It is structurally very similar to NAD+, but it contains an extra phosphate group and plays an important role in anabolic reactions, such as photosynthesis. Another nucleotide-based electron carrier that you will also encounter in this course and beyond, flavin adenine dinucleotide (FAD+), is derived from vitamin B2, also called riboflavin. Its reduced form is FADH2. Learn to recognize these molecules as electron carriers as well.

    NAD.jpg

    Figure 1. The oxidized form of the electron carrier (NAD+) is shown on the left, and the reduced form (NADH) is shown on the right. The nitrogenous base in NADH has one more hydrogen ion and two more electrons than in NAD+.

    NAD+ is used by the cell to "pull" electrons off of compounds and to "carry" them to other locations within the cell; thus it is called an electron carrier. NAD+/H compounds are used in many of the metabolic processes we will discuss in this class. For example, in its oxidized form, NAD+ is used as a reactant in glycolysis and the TCA cycle, whereas in its reduced form (NADH), it is a reactant in fermentation reactions and the electron transport chain (ETC). Each of these processes will be discussed in later modules.

    Energy story for a red/ox reaction

    ***As a rule of thumb, when we see NAD+/H as a reactant or product, we know we are looking at a red/ox reaction.***

    When NADH is a product and NAD+ is a reactant, we know that NAD+ has become reduced (forming NADH); therefore, the other reactant must have been the electron donor and become oxidized. The reverse is also true. If NADH has become NAD+, then the other reactant must have gained the electron from NADH and become reduced.

    NAD2.jpg

    Figure 2. This reaction shows the conversion of pyruvate to lactic acid coupled with the conversion of NADH to NAD+. Source: https://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/sequential_reactions

    In the figure above, we see pyruvate becoming lactic acid, coupled with the conversion of NADH into NAD+. This reaction is catalyzed by LDH. Using our "rule of thumb" above, we categorize this reaction as a red/ox reaction. NADH is the reduced form of the electron carrier, and NADH is converted into NAD+. This half of the reaction results in the oxidation of the electron carrier. Pyruvate is converted into lactic acid in this reaction. Both of these sugars are negatively charged, so it would be difficult to see which compound is more reduced using the charges of the compounds. However, we know that pyruvate has become reduced to form lactic acid, because this conversion is coupled to the oxidation of NADH into NAD+. But how can we tell that lactic acid is more reduced than pyruvate? The answer is to look at the carbon-hydrogen bonds in both compounds. As electrons are transferred, they are often accompanied by a hydrogen atom. There is a total of three C-H bonds in pyruvate, and there is a total of four C-H bonds in lactic acid. When we compare these two compounds in the before and after states, we see that lactic acid has one more C-H bond; therefore, lactic acid is more reduced than pyruvate. This holds true for multiple compounds. For example, in the figure below, you should be able to rank the compounds from most to least reduced using the C-H bonds as your guide.

    ex1.jpg

    Figure 3. Above are a series of compounds than can be ranked or reorganized from most to least reduced. Compare the number of C-H bonds in each compound. Carbon dioxide has no C-H bonds and is the most oxidized form of carbon we will discuss in this class. Answer: the most reduced is methane (compound 3), then methanol (4), formaldehyde (1), carboxylic acid (2), and finally carbon dioxide (5).

    NADpro.jpg

    Figure 4. This reaction shows the conversion of G3P, NAD+, and Pi into NADH and 1,3-BPG. This reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

    Energy story for the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase:

    Lets make an energy story for the reaction above.

    First, lets characterize the reactants and products. The reactants are glyceraldehyde-3-phosphate (a carbon compound), Pi (inorganic phosphate), and NAD+. These three reactants enter into a chemical reaction to produce two products, NADH and 1,3-bisphosphoglycerate. If you look closely, you can see that the 1,3-BPG contains two phosphates. This is important when we are double checking that no mass has been lost. There are two phosphates in the reactants, so there must be two phosphates in the products (conservation of mass!). You can double check that all the other atoms are also accounted for. The enzyme that catalyzes this reaction is called glyceraldehyde-3-phosphate dehydrogenase. The standard free energy change of this reaction is ~6.3 kJ/mol, so under standard conditions, we can say that the free energy of the products is higher than that of the reactants and that this reaction is not spontaneous under standard conditions.

    What can we say about this reaction when it is catalyzed by glyceraldehyde-3-phosphate dehydrogenase?

    This is a red/ox reaction. We know that because we have produced a reduced electron carrier (NADH) as a product and NAD+ is a reactant. Where did the electron come from to make NADH? The electron must have come from the other reactant (the carbon compound).

    Note: recommended discussion

    We will spend some time examining the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase in more detail as we move through the lectures and text. The first thing to discuss here is that the figure above is a highly simplified or condensed version of the steps that take place—one could in fact break that reaction above into TWO conceptual reactions. Can you imagine what those two "subreactions" might be? Discuss amongst yourselves.

    Note: recommended discussion

    The text above notes that the standard change in free energy for this complex reaction is ~+6.3 kJ/mol. Under standard conditions, this reaction is NOT spontaneous. However, this is one of the key reactions in the oxidation of glucose. It needs to GO in the cell. The questions are as follows: why is it important to note things like "standard change of free energy" or "under standard conditions" when reporting that ΔG°? What could possibly be going on in the cell to make what is under standard conditions an endergonic reaction "go"?

    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 be expected 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).

    ATP.jpg


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

    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 free energy of a reaction between the products and reactants, ΔG. In this case we are explicitly assigning direction to the reaction, either in the direction of 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. Some free energy can be associated 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. A similar analysis could be made with the reverse reaction.

    Possible Exercise

    Use the figure of ATP above and your knowledge of what a water molecule looks like to draw a figure of the reaction steps described above: breaking of the phosphoanhydride bond, breaking of the water, and formation of new bonds to form ADP and inorganic phosphate. Track the atoms in different colors if that helps.

    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 does not only have 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.

    03highnrg.png


    Figure 2. The 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

    table_hydrolysis.png


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

    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

    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.

    splash_ATP.png

    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, it is hypothesized 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). Our study of glycolysis will therefore be examined using the precepts outlined in the energy challenge rubric that ask us to formally consider what happens to BOTH matter and energy in this multistep 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 actually evolved, and thinking about alternative strategies for satisfying various constraints will come in handy then.

    In the context of the energy story, we will ask you to think about glycolysis as a process from which something can be learned by analyzing what happens to both matter and energy. That is, even though it is a ten-step biochemical pathway, we propose that some insight can be learned by carefully examining the process as a set of matter and energy inputs and outputs, a process with a beginning and an end.

    So what is glycolysis? Let's start to find out.

    glycolysis_2panel.png

    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)

    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 than ΔG°' measurements due to cellular conditions, such as concentrations of relevant metabolites etc. There are three large, negative ΔG drops in the cell in the process of glycolysis. These reactions are considered 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

    Overall, the glycolytic pathway consists of 10 enzyme-catalyzed steps. The primary input into this pathway is a single molecule of glucose, though we will discover that molecules may feed in and out of 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

    The first few steps of glycolysis are typically referred to 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 of the 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.

    glycolysis_first_half.png

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

    Suggested discussion

    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), does this give you 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 effectively tags it for several different possible fates (see Figure 3).

    fates_of_g6p.png

    Figure 3. Note that this figure indicates 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)

    As Figure 3 indicates, glycolysis is but one possible fate for glucose 6-phosphate (G6P). Depending on cellular conditions, G6P may be diverted to the biosynthesis of glycogen (a form of 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.

    Suggested discussion

    We discussed allosteric regulation of an enzyme in earlier modules but did so in a context where the enzyme was "alone." Now let's consider the enzyme in the context of an extended metabolic pathway(s). Can you now express why allosteric regulation is functionally important and how it can be used to regulate the flow of compounds through a pathway? Try to express yourself.

    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 thus 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.

    18323310-q-07-650-a542d8629a-1478168682.png

    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).

    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 types of chemical reactions that we've studied so far. If you're energy focused, this is finally a step of glycolysis where some of the reduced sugar is oxidized. The reaction is catalyzed by the enzyme glyceraldehyde 3-phosphate dehydrogenase. This enzyme catalyzes a multistep reaction between three substrates—glyceraldehyde 3-phosphate, the cofactor NAD+, and inorganic phosphate (Pi)—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. In this particular case, the red/ox reaction, a transfer of electrons off of G3P and onto NAD+, is exergonic, and the phosphate transfer happens to be 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 multistep 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 be continuously oxidized back into NAD+ in order to keep this step going. If NAD+ is not available, the second half of glycolysis slows down or stops.

    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.

    Possible discussion

    If a transfer of a phosphate from 1,3-BPG to ADP is exergonic, what does that say about the free energy of hydrolysis of the phosphate from 1,3-BPG as compared to the free energy of hydrolysis of the terminal phosphate on ATP?

    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 is run 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 NAD+ is not regenerated, all of the cell's NAD will be nearly completely transformed into NADH. So how do cells regenerate NAD+?

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

    Strongly suggested discussion/exercise

    Can you write an energy story for the overall process of glycolysis? For energy terms, just worry about describing things in terms of whether they are exergonic or endergonic. When I say "overall process," I mean overall process: glucose should be listed on the reactant side of the arrow, and pyruvate should be listed on the product side of the arrow.

    Substrate-level phosphorylation (SLP)

    The simplest route to synthesize ATP is substrate-level phosphorylation. ATP molecules are generated (that is, regenerated from ADP) as a direct result 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. It can be found in a variety of catabolic reactions, most notably in two specific reactions in glycolysis (which we will discuss specifically later). Suffice it to say that what is required is a high-energy intermediate whose oxidation is sufficient to drive the synthesis of ATP.

    sub.jpg

    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)

    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).


    SS1_2018_Lecture_05 is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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