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Metabolic Regulation and the Endosymbiotic Theory

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You should have a working knowledge of the following terms:

  • activation energy
  • active site
  • allosteric enzyme
  • allosteric regulation
  • catalyst
  • coenzyme
  • cyanobacteria
  • endosymbiosis
  • enzyme
  • enzyme-substrate complex
  • extant
  • feedback inhibition (negative feedback)
  • feedback stimulation (positive feedback)
  • inhibitor
  • kinase
  • phosphofructokinase
  • serial endosymbiosis
  • substrate

Introduction and Goals

This tutorial will explore metabolic regulation by focusing on the glycolytic component of cellular respiration. Although all of the metabolic processes that occur in cells are regulated, cellular respiration especially provides excellent examples of regulatory mechanisms. You will learn some basics about how enzymes control metabolism, and then look more closely at the enzymatic regulation of glycolysis. You will also learn more about mitochondria (the organelles where much of cellular respiration takes place) and some current thoughts about their origins. By the end of this tutorial you should be familiar with:

  • The mechanisms of allosteric regulation
  • The mechanisms of feedback inhibition
  • The inhibition and stimulation of enzyme activity
  • The enzymatic control of cellular metabolism
  • The endosymbiont theory of the origin of mitochondria
  • The selective pressures that might have favored the acquisition of mitochondria

Overview of Cellular Respiration

Before we discuss the regulation of cellular respiration, the entire process will be reviewed. This figure provides a good review of the process. Be sure that you understand where the main reactions occur within a eukaryotic cell and how the various components work together to insure optimal transduction of energy. Recall, cellular respiration involves glycolysis, the Krebs cycle, and the electron transport chain. Glycolysis, which takes place in the cytosol of cells, breaks down single molecules of glucose into twin molecules of the compound pyruvate. In addition, a small amount of ATP (the energetic currency of cellular metabolism) is released in this process. A catabolite of pyruvate, acetyl CoA, is fed into the Krebs cycle. The Krebs cycle is a collection of enzymes located in the mitochondrion (plural, mitochondria). These enzymes convert acetyl CoA further (again, producing a small amount of ATP directly) and generate the highly reduced compounds NADH and FADH2, which, in turn, send their electrons through the electron transport chain in the mitochondrial membrane (where they are typically donated eventually to oxygen). The energy generated by the flow of electrons causes the buildup of a hydrogen gradient across this membrane, representing an enormous store of potential energy in the form of a charge differential (voltage). The charge differential is dissipated when hydrogen ions flow through the membrane via molecules of ATP synthase, which use their energy to form a relatively large amount of ATP by oxidative phosphorylation.

Figure 1.  An Overview of Cellular Respiration.  (Click to enlarge)

Catalysis I - Activation Energy

How is cellular respiration regulated? For that matter, how is any biochemical process regulated? Must cells wait for reactions to occur spontaneously? Recall, chemical reactions that release free energy are called exergonic reactions. Exergonic reactions release energy, whereas endergonic reactions require (absorb) energy.

Every working cell depends on thousands of exergonic reactions that, by definition, occur spontaneously. Do not confuse spontaneity with instantaneous. In fact, many spontaneous reactions take a long time to occur. This is because some activation energy is required to start a reaction. This figure shows the path of an exergonic reaction. Importantly, note that the free energy of the reaction rises before it falls at the completion of the reaction.

Figure 2. Spontaneous reactions require an input of energy. (Click to enlarge)

Even exergonic reactions require some input of energy, which is referred to as activation energy. This energy is needed to break bonds in the reactant molecules, then even more energy is released (in an exergonic reaction) when the bonds in the product molecules form. Some reactions have activation energy barriers that are sufficiently low enough to allow them to take place at room temperature, but most reactions require the input of more energy before they will occur. This is not an abstract concept. The oxidation of wood by oxygen (during burning) is a highly exergonic reaction, and will occur spontaneously. Luckily for those of us living in wood houses, a high activation energy is required for this reaction to occur.

Cells must overcome numerous activation energy barriers in order to carry out metabolic processes. This is not done by applying extra energy to reactions to speed them up, but by employing protein catalysts called enzymes to facilitate reactions. Enzymes do not add energy; instead, they hasten reactions by lowering the activation energy necessary so that the reactions can occur at normal metabolic temperatures. This figure shows the energetic relationship between an enzymatically catalyzed reaction and one that is not catalyzed.

Catalysis II - The Enzyme-Substrate Complex

The three-dimensional shape of an enzyme renders it highly specific and allows it to act only on a certain type of molecule, its substrate. Substrates fit into the active site of an enzyme, much like a key fits into a lock. As shown in this figure, a substrate binds to an enzyme's active site, where it is held in a position that initially promotes the breaking of chemical bonds, and later the formation of new bonds associated with the product. The unit that forms when an enzyme and its substrate(s) join is the enzyme-substrate complex. Products are made and released after the enzyme-substrate complex forms, and afterwards the enzyme emerges unchanged from the reaction. Most importantly, the activation energy required for the catalyzed reaction was decreased.

Figure 3. Enzymes are highly specific in their action.  (Click to enlarge) 

The sugar contained in sport drinks is most likely sucrose. Sucrose (a disaccharide) is broken down into the smaller sugars (monosaccharides) glucose and fructose by the enzyme sucrase. Sucrase has only one role in the cell. For example, it is not capable of switching functions and breaking down the sugar lactose. The fit between an enzyme and its substrate is very specific, and each enzyme can catalyze only one kind of reaction involving a specific substrate or set of substrates. Therefore, thousands of different enzymes can be required to perform all of a cell's metabolic processes.

There are enzymes that catalyze just about every reaction you can think of. There are even companies that specialize in putting enzymes to industrial use. Visit Novozymes for an example of such a company.

Metabolic Regulation I - Coenzymes

Enzymatic activity is influenced by many things that are not mutually exclusive. For example, temperature and pH can have profound effects on enzymes. Increases in temperature tend to increase reaction rates until a point is reached at which the bonds stabilizing the three-dimensional structure of an enzyme start to degrade, hence denaturing the protein and disrupting the conformation of its active site. Likewise, changes in pH can affect the stability of chemical bonds and the integrity of active sites.

In many cases, enzymes must be accompanied by coenzymes, which are not themselves catalysts but are required for normal catalytic functioning of the enzyme. If cells are deficient in certain coenzymes, their associated enzymes will not work properly. The vitamins and minerals that are essential in our diets are often coenzymes or they are the raw materials required to manufacture these important compounds. One such substance, coenzyme Q10, has received much attention recently for its role as a powerful antioxidant and its potential as a treatment for cancer (though much of this attention is still based on speculation).

One of the most common forms of metabolic regulation is feedback inhibition, whereby a reaction's end products inhibit the reaction itself by halting it when a certain amount of product has accumulated. Feedback inhibition is accomplished in two ways: via allosteric regulation (noncompetitive inhibition), whereby an inhibitor binds to a site distant from the active site and causes a conformational change in the enzyme thereby decreasing its ability to bind with the substrate and hence catalyze the reaction, or via competitive inhibition where the inhibitor binds to the active site, directly preventing the formation of an enzyme-substrate complex. Allosteric enzymes are generally intricate molecules, composed of at least two protein subunits; conformational changes occur when allosteric sites are filled, thereby changing the shape of the active site itself.

Allosteric regulation can involve stimulation of enzyme activity and feedback inhibition. Certain allosteric sites bind activators, which leads to changes that stabilize an enzyme's active site, and thus increase its affinity for substrate. This figure illustrates how allosteric enzymes can be either activated or inhibited. Note the importance of the 3-D conformation in both processes; activators serve to expose the enzyme's active site, whereas inhibitors change the enzyme's conformation so that the substrate cannot bind.

Figure 4.  Enzyme Substrate and Binding Inhibition.  (Click to enlarge)

Glycolysis is a Dynamic Process

As you've learned, glycolysis converts glucose to pyruvate through a series of steps. Each step has its own enzyme that catalyses the particular reaction.

Phosphofructokinase (PFK) is an important enzyme that is involved in the early stages of glycolysis. It belongs to a family of enzymes known as kinases. Kinases move phosphate groups from energy-containing molecules (e.g., ATP) to other organic molecules.

Phosphofructokinase removes a phosphate from ATP and transfers it to an intermediate product, fructose 6-phosphate, thereby transforming this compound into the more symmetrical molecule fructose 1, 6-bisphosphate. Subsequent reactions, catalyzed by different enzymes, cleave the new molecule in two and ultimately produce two pyruvate molecules (one for each half of the fructose 1,6-bisphosphate) as the glycolytic end product. Phosphofructokinase is a critical enzyme in the glycolytic pathway because it represents the first point of metabolic regulation.

You should recognize that phosphofructokinase is an allosteric enzyme. That is, it has allosteric sites that bind inhibitors and activators, influencing the affinity of the active site for the substrate fructose 6-phosphate. As shown in this figure, ATP and citrate inhibit the activity of this enzyme, and thus slow the progression of glycolysis. Conversely, AMP (adenosine monophosphate) stimulates phosphofructokinase activity and sets glycolysis in motion. The figure below shows the relationship between the overall shape of the phosphofructokinase enzyme, the location of the substrate binding site (central), and the inhibitor/activation sites (left/right).

 Figure 5.  The Dynamics of Glycolysis. (Click to enlarge)


Figure 6.  The enzyme Phosphofructokinase (PFK).

Why Regulate Glycolysis?

Why would an organism need to regulate glycolysis? Or, put another way, what would be the selective advantage of regulating this process? Glycolysis is the first component of cellular respiration; it produces raw materials for the Krebs cycle, and ultimately the electron transport chain (which operates to provide cells with the ATP they need for metabolic processes). ATP and the compounds required for its synthesis are of great importance, but cells would not be operating at peak efficiency if they produced pyruvate constantly. During times of low activity, energy requirements are less, and therefore there is no need to divert valuable organic molecules into either ATP or the intermediates of cellular respiration. In other words, when ATP levels are high, there is little need for pyruvate; therefore, glycolysis can be restricted for the time being; accordingly, high ATP levels inhibit phosphofructokinase. The activity of this enzyme is also inhibited by citrate, a product of the Krebs cycle. This allows cells to coordinate the rates of glycolysis and the Krebs cycle, slowing down pyruvate production if the Krebs cycle gets too far ahead.

Conversely, high levels of AMP stimulate phosphofructokinase activity. When ATP is low, ADP is inevitably high, and therefore some of this ADP is converted into AMP. High levels of AMP indicate that ATP is in short supply and that it needs to be regenerated. By "turning on" glycolysis, the cell can increase the production of compounds that feed into the Krebs cycle and the electron transport chain, thus elevating the rate of ATP synthesis.

Metabolically, glycolysis is an open system. This means that molecules can enter or leave at any point. Enzymatic pathways are not simple linear progressions; they are dynamic processes that are typically regulated at many intermediate points, depending on the immediate needs of the cell.

The Localization of Enzymes

 Figure 7.  Cellular metabolism.  (Click to enlarge)

This diagram depicts only a fraction of the metabolic pathways in a cell. Dots represent molecules, and lines represent chemical reactions.
There are thousands of enzymes in most eukaryotic cells. This figure illustrates the various pathways that interconnect cellular respiration with other reactions. The compartmentalization of enzymes increases the efficiency of reactions and prevents enzymes from getting lost in the shuffle of cellular metabolism. For example, the helicases that unwind the double helix of DNA, in preparation for replication, are concentrated in the nucleus of eukaryotic cells, where they are utilized during the replication process. If they simply diffused freely throughout the cell and if replication depended on rare chance encounters between double helices and helicases, replication would occur with far less efficiency.

Similarly, many of the enzymes involved in cellular respiration are concentrated at specific sites within cells. For example, while glycolytic enzymes are found in the cytosol, the enzymes involved in the Krebs cycle are located within the mitochondrial matrix. Enzymes of the electron transport chain are actually imbedded within the inner membrane of mitochondria, where they are clustered in functional groups, allowing reactions to take place in sequence.

Conversely, high levels of AMP stimulate phosphofructokinase activity. When ATP is low, ADP is inevitably high, and therefore some of this ADP is converted into AMP. High levels of AMP indicate that ATP is in short supply and that it needs to be regenerated. By "turning on" glycolysis, the cell can increase the production of compounds that feed into the Krebs cycle and the electron transport chain, thus elevating the rate of ATP synthesis.

Metabolically, glycolysis is an open system. This means that molecules can enter or leave at any point. Enzymatic pathways are not simple linear progressions; they are dynamic processes that are typically regulated at many intermediate points, depending on the immediate needs of the cell.

The Origins of Eukaryotic Metabolic Complexity

Metabolic regulation is sophisticated and intricate, controlled in fine detail by enzymes. These catalysts oversee cellular respiration, along with an overwhelming majority of metabolic processes in prokaryotes and eukaryotes. The automation, integration, and incredible complexity of enzymatically driven reactions facilitate life. They regulate events like the transformation of soil nutrients and solar energy into plant tissue, which can then go into the flesh of animals like this Thompson's gazelle, and finally, into predators like this hunting cheetah.

Figure 8.  Predator-prey relationships are metabolically regulated. (Click to enlarge)

Figure 9. Rational Thought is Metabolically Regulated. (Click to enlarge)

The organization orchestrated by enzymes also bestows upon higher organisms (e.g., humans) the capacity for rational thought.

Where did this sophisticated organization come from? How is it that life, in particular eukaryotic life, was able to evolve such a diverse and astonishing array of finely tuned metabolic functions? One possibility is that the acquisition of mitochondria by eukaryotes allowed the efficient production of large quantities of ATP, thus making it possible for emerging lineages of eukaryotic organisms to support elaborate functions that require energy (e.g., motility).

Next we will discuss mitochondrial structure and current thoughts about mitochondrial evolution.

The Mitochondrion

Mitochondria are present in the cells of most eukaryotes. As you've learned, they are a site of ATP synthesis. They are also quite abundant; some cells contain a single large mitochondrion, but most eukaryotic cells contain many more. Cells that require a great deal of energy (e.g., muscle cells) can house thousands of mitochondria. The basic structure of a mitochondrion is depicted here. Mitochondria are enclosed in a double membrane; a smooth outer membrane and an inner membrane that is contorted into a complex of infoldings called cristae. Cristae provide extensive surface areas for processes such as the electron transport chain, which takes place within the inner membrane. This membrane typically contains thousands of copies of electron transport chain proteins. The space within the cristae is the mitochondrial matrix. Recall, this is where the Krebs cycle occurs.

Figure 10. The structure of a mitochondrion.  (Click to enlarge)  

Mitochondria also possess their own genetic material in the form of circular DNA (which, as you will learn, provides an important clue as to how mitochondria arose). Mitochondria and their genetic material are maternally inherited in sexually reproducing organisms. (The sperm mitochondria rarely enter the egg during fertilization.) Therefore, mitochondria are passed to offspring in the cytoplasm of ova. Hence, your mitochondria are virtually identical to your mother's. There are many fascinating consequences associated with this phenomenon. For example, a number of maternally inherited diseases are caused by defective genes in mitochondria.

For a close-up look at mitochondria (plus chloroplasts and a nucleus), see the Virtual Cell. Select "mitochondrion" or "cristae" in the pull-down menu and click on the image. Keep clicking or use the buttons on the right to navigate through the cell. If you are having trouble, select "About Virtual Cell" for information on how to explore the site. Be prepared to answer some questions about the organization of mitochondria when you return, with special reference to where the electron transport chain is located.

The Endosymbiont Theory

Figure 11. Endosymbiosis. (Click to enlarge)  A typical eukaryotic cell contains a variety of organelles, some of which are of prokaryotic origin.

It is thought that mitochondria evolved from prokaryotes that inhabited the cells of other larger prokaryotes. This scenario likely originated as an endosymbiosis, in which one organism began living within the body of another. (This type of association is very common.) Put simply, it is thought that a relatively large prokaryote (a protoeukaryote) engulfed a smaller prokaryote (a protomitochondrion), and instead of the larger consuming the smaller, they formed an everlasting relationship. This engulfment would account for the existence of the inner membrane of mitochondria (the ancestral prokaryotic membrane) and the outer membrane. The symbiosis was mutualistic because it benefited both the protoeukaryote and the protomitochondrion; the protomitochondrion was provided with a safe environment and plenty of raw materials for respiration, and the protoeukaryote was provided with a rich, free supply of fuel in the form of ATP and some safety from the oxidizing power of O2; to be discussed in the next section.

Chloroplasts, which are found in eukaryotic photosynthetic cells, might also have originated from an ancient symbiosis. In the case of chloroplasts, however, the prokaryote was photosynthetic (probably an ancient cyanobacterium; discussed later in this tutorial). In this case, the ancestors to the modern cells that possess both mitochondria and chloroplasts, at some point in the past, underwent a serial endosymbiosis (sequential endosymbiotic events).

Evidence for the Endosymbiont Theory

Evidence for the endosymbiont theory is strong. Most convincingly, mitochondria closely resemble extant (currently existing) bacteria. Mitochondria are similar in size, and they replicate in a fashion that is very similar to binary fission. Also, the inner membrane of mitochondria bears a strong resemblance to the membranes of prokaryotes, sharing several key proteins and transport systems. Additionally, mitochondria have their own DNA (which takes the form of a circular plasmid, like that of prokaryotes) and they possess all of the cellular machinery required to transcribe and translate their genomes, thereby enabling them to produce their own proteins.

The character of mitochondrial DNA is much like that seen in modern-day prokaryotes. For example, mitochondrial DNA lack histones, which are associated with eukaryotic nuclear DNA but not with prokaryotic DNA. Additionally, mitochondrial ribosomes are more similar in behavior, structure, and nucleic acid base sequence to the ribosomes of prokaryotes than they are to eukaryotic ribosomes. For example, there is high sequence similarity between the ribosomal RNA of mitochondria and that of modern endosymbiotic bacteria.

The relationship between mitochondria and eukaryotes has grown so intimate that neither can exist without the other. Most mitochondrial genes are contained within the nucleus of their eukaryotic host cells, which makes mitochondria unable to reproduce and survive independently. Likewise, eukaryotes (e.g., humans) would be unable to manufacture enough ATP to sustain life without the help of mitochondria.

Selective Pressures Favoring Symbiosis

Sometime before 2.2-2.3 billion years ago, cyanobacteria evolved the ability to use H2O and CO2 to make organic molecules with the help of solar energy, in a process known as photosynthesis. The primary "waste" product from this type of photosynthesis is O2, which began to accumulate in the atmosphere due to the activity of cyanobacteria. Other earlier forms of photosynthesis probably relied on H2S rather than H2O and did not result in the release of O2. For millions of years, cyanobacterial photosynthesis did not change the Earth's atmosphere; oxygen released by photosynthesizing mats of marine cyanobacteria combined with iron ions in the ocean, forming iron oxide that precipitated to the sea floor. When these iron ions were depleted, oxygen began to accumulate in seawater and eventually it diffused into the atmosphere.

Figure 12.   A Cyanobacteria.  This blue-green alga is one type of cyanobacteria.

Figure 13.  An Algal Bloom. (Click to enlarge)  This pond is located in San Francisco's Japanese Tea Garden in Golden Gate Park.

The change in atmospheric composition due to cyanobacteria was enormous, even more severe than the pollution associated with industrialized civilization. Oxygen is a powerful oxidizer, and its tendency to strip electrons and attack the bonds of organic molecules can be very dangerous to living organisms. The atmospheric changes effected by cyanobacteria probably resulted in numerous extinctions, but conversely, they also led to novel adaptations (e.g., the production of antioxidants). Furthermore, some organisms also evolved the ability to detoxify oxygen by reducing it with electrons from other molecules. Electron transport chains, such as the ones that exist in modern-day mitochondria, might have evolved as mechanisms for counteracting the destructive effects of O2, secondarily becoming a means of energy production over evolutionary time. The ancestors of eukaryotes might have gained an advantage in an oxygen-rich atmosphere by adopting endosymbionts to detoxify O2. The additional benefit that eukaryotes enjoy today, ATP synthesis, might be a derived function.


This tutorial addressed the regulation of metabolism. Metabolism is regulated at a number of steps. Consider the enzyme phosphofructokinase, which is not always active. Cellular control is obtained via a feedback inhibition, by which high levels of ATP (or citrate) lead to a decrease in the catalytic activity of the enzyme. This form of control means that ATP production only occurs when the level of ATP (or citrate) falls below a certain level. This mechanism helps the cell produce only that amount of ATP needed at any given time. Conversely, as ATP usage goes up (and ATP levels fall), the enzyme works more effectively, allowing more ATP to be produced.

In most eukaryotic cells, the majority of ATP is produced in the mitochondria. Eukaryotic cells are more complex than prokaryotic cells, and this complexity is largely due to the appearance of discrete membrane-bound compartments in which various cellular processes are compartmentalized. In prokaryotes, most cellular processes occur within one compartment.

The available data indicate that two major evolutionary processes have contributed to the complexity of eukaryotic cells. First, it appears that the plasma membrane has undergone various modifications. These invaginations have resulted in complex intracellular compartments such as lysosomes, the endoplasmic reticulum, and golgi apparatuses. The majority of DNA is found within a membrane-bound nucleus. Second, the mitochondria seem to be of prokaryotic origin. There is compelling data to indicate that during the early evolution of eukaryotes, an intimate association occurred between a primitive prokaryote and a primitive eukaryote. This intracellular symbiosis became permanent over the course of time, and what exists today is a stable remnant of that ancient association.

Here is a quote by Lewis Thomas from his classic book of essays, The Lives of a Cell, which puts these facts in a provocative context:

A good case can be made for our nonexistence as entities. We are not made up, as we had always supposed, of successively enriched packets of our own parts. We are shared, rented, occupied. At the interior of our cells, driving them, providing the oxidative energy that sends us out for the improvement of each shining day, are the mitochondria, and in a strict sense, they are not ours. They turn out to be little separate creatures, the colonial posterity of migrant prokaryocytes, probably primitive bacteria that swam into ancestral precursors of our eukaryotic cells and stayed there. Ever since, they have maintained themselves and their ways, replicating in their own fashion, privately, with their own DNA and RNA quite different from ours. They are as much symbionts as the rhizobial bacteria in the roots of beans. Without them, we would not move a muscle, drum a finger, think a thought....