Bis2A_Singer_Glycolysis
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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;
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
So what is
| 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 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.
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)
The paragraph above states that the enzyme hexokinase has "broad specificity." This means that it can
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).
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)
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.
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 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 (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. 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.
Possible NB Discussion 
Point
Can you write an energy story for Step 6 of glycolysis (the reaction
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?
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.
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.
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:
Pyruvate + NADH ↔ lactic acid + NAD+
Figure 1. Lactic acid fermentation converts pyruvate (a slightly oxidized carbon compound) to lactic acid. In the process, NADH is oxidized to form NAD+. Attribution: Marc T. Facciotti (original work)
Energy story for the fermentation of pyruvate to lactate
An example (if a bit lengthy) energy story for lactic acid fermentation is the following:
The reactants are pyruvate, NADH, and a proton. The products are lactate and NAD+. The process of fermentation results in the reduction of pyruvate to form lactic acid and the oxidation of NADH to form NAD+. Electrons from NADH and a proton are used to reduce pyruvate into lactate. If we examine a table of standard reduction potential, we see under standard conditions that a transfer of electrons from NADH to pyruvate to form lactate is exergonic and thus thermodynamically spontaneous. The reduction and oxidation steps of the reaction are coupled and catalyzed by the enzyme lactate dehydrogenase.
A second example: alcohol fermentation
Another familiar fermentation process is alcohol fermentation, which produces ethanol, an alcohol. The alcohol fermentation reaction is the following:
Figure 2. Ethanol fermentation is a two-step process. Pyruvate (pyruvic acid) is first converted into carbon dioxide and acetaldehyde. The second step converts acetaldehyde to ethanol and oxidizes NADH to NAD+. Attribution: Marc T. Facciotti (original work)
In the first reaction, a carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas (some of you may be familiar with this as a key component of various beverages). The second reaction removes electrons from NADH, forming NAD+ and producing ethanol (another familiar compound—usually in the same beverage) from the acetaldehyde, which accepts the electrons.
Fermentation pathways are numerous
While the lactic acid fermentation and alcohol fermentation pathways described above are examples, there are many more reactions (too numerous to go over) that Nature has evolved to complete the NADH/NAD+ cycle. It is important that you understand the general concepts behind these reactions. In general, cells try to maintain a balance or constant ratio between NADH and NAD+; when this ratio becomes unbalanced, the cell compensates by modulating other reactions to compensate. The only requirement for a fermentation reaction is that it uses a small organic compound as an electron acceptor for NADH and regenerates NAD+. Other familiar fermentation reactions include ethanol fermentation (as in beer and bread), propionic fermentation (it's what makes the holes in Swiss cheese), and malolactic fermentation (it's what gives Chardonnay its more mellow flavor—the more conversion of malate to lactate, the softer the wine). In Figure 3, you can see a large variety of fermentation reactions that various bacteria use to reoxidize NADH to NAD+. All of these reactions start with pyruvate or a derivative of pyruvate metabolism, such as oxaloacetate or formate. Pyruvate is produced from the oxidation of sugars (glucose or ribose) or other small, reduced organic molecules. It should also be noted that other compounds can be used as fermentation substrates besides pyruvate and its derivatives. These include methane fermentation, sulfide fermentation, or the fermentation of nitrogenous compounds such as amino acids. You are not expected to memorize all of these pathways. You are, however, expected to recognize a pathway that returns electrons to products of the compounds that were originally oxidized to recycle the NAD+/NADH pool and to associate that process with fermentation.
Figure 3. This figure shows various fermentation pathways using pyruvate as the initial substrate. In the figure, pyruvate is reduced to a variety of products via different and sometimes multistep (dashed arrows represent possible multistep processes) reactions. All details are deliberately not shown. The key point is to appreciate that fermentation is a broad term not solely associated with the conversion of pyruvate to lactic acid or ethanol. Source: Marc T. Facciotti (original work)
A note on the link between substrate-level phosphorylation and fermentation
Fermentation occurs in the absence of molecular oxygen (O2). It is an anaerobic process. Notice there is no O2 in any of the fermentation reactions shown above. Many of these reactions are quite ancient, hypothesized to be some of the first energy-generating metabolic reactions to evolve. This makes sense if we consider the following:
- The early atmosphere was highly reduced, with little molecular oxygen readily available.
- Small, highly reduced organic molecules were relatively available, arising from a variety of chemical reactions.
- 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.
- The process evolved long before O2 was found in the environment.
- The substrates, highly reduced, small organic molecules, like glucose, were readily available.
- The end products of many fermentation reactions are small organic acids, produced by the oxidation of the initial substrate.
- 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.
- This suggests that substrate-level phosphorylation and fermentation reactions coevolved.