Energy Metabolism I: _{Glycolosis and the Krebs cycle}

Breaking down fuel for cellular energy

In many cases, the assembly line idea seemed to be the only one that made sense conceptually. Consider glucose, for example, commonly known as blood sugar. (See the structure of a glucose molecule in Figure 2.) By the turn of the 20^th century, scientists knew that glucose was one of the main fuels, or sources of energy, for animals, bacteria, and yeast. Setting glucose on fire in the laboratory produced carbon dioxide (CO₂) and water (H₂O), the same compounds that animals produced when they exercised. However, no one believed that cells could have tiny fires inside. Observations under the microscope certainly did not show any flames. Nevertheless, people do feel a burning sensation in their muscles during heavy exercise.

Realizing that that the breakdown of body fuels probably took place in a controlled series of steps, researchers imagined enzymes working like factory workers, modifying different parts of a particular chemical compound. Like the workers on an assembly line, each enzyme would make one special change to each molecule. The altered molecule would then be further modified, step by step by different enzymes, and this could happen not only during the breakdown of fuels; it also could happen during the production, or synthesis, of needed biological molecules using simpler chemicals as building material.

Glucose and other sugars belong to the class of macromolecules called carbohydrates (see our Carbohydrates module). Along with lipids and proteins, carbohydrates play a variety of roles in organisms, and one role is providing cells with energy. While glucose and fats (a class of lipid) are the preferred energy compounds, proteins also can be used as fuel (see our modules Lipids and Fats and Proteins to learn more). Like the logs of a cabin, proteins are made from building blocks called amino acids, which can be used in multiple ways. They can be put together giving the cabin structure, but if needed they can also be burned as firewood to keep the cabin warm.

Within structures called mitochondria, microscopic power plants in the cells of eukaryotes (Figure 3), broken down bits of carbohydrates, fats, and proteins all come together, feeding into a kind of reverse assembly line that goes around and around in a cycle. As the cycle goes around, the various energy-rich bits are incorporated at different stations. At the same time, the cycle sends other products away to other areas of the power plant. The pathway has many names, including the citric acid cycle and the tricarboxylic acid cycle (TCA), because of the compounds that cycle within it. However, it’s also known as the Krebs cycle, for its discoverer Sir Hans Adolf Krebs.

Cyclic assembly lines

Born August 25, 1900, in Germany, Krebs earned his MD and began his research career working with Otto Heinrich Warburg. A pioneer in biochemistry, Warburg was the inventor of the manometer, an instrument that could measure oxygen and other gasses in blood and other fluids. Warburg was one of the lead biochemists worldwide, and in the early 20th century his country was the best place for emerging researchers like Krebs to get an education. Germany in this era was the global center of scientific research, especially in all areas of chemistry. So frequent were German publications in research journals that students aspiring to science worldwide would learn German just to be prepared to read the new articles. This was the world in which Krebs came of age.

Krebs discovers the urea cycle

Using the Warburg manometer, Krebs made his first big discovery, the urea cycle (also called the ornithine cycle). By the late 1920s, it was well know that the breakdown of amino acids in animals must release ammonia (NH₃). Krebs new that ammonia is toxic, yet somehow the body is able to convert it to urea, a chemical that is easily excreted in urine. Thinking assembly line style, Krebs and his student, Kurt Henseleit, came up with a hypothetical set of reactions, beginning with the conversion of ornithine into another chemical by receiving a piece of the amino acids containing the ammonia. The manometer allowed Krebs to analyze samples of animal liver exposed to the intermediary chemicals that they suspected were made from ornithine. Krebs and Henseleit, were able to test and tweak their hypothesis, reaction by reaction. The pathway of reactions was a cycle, because, after a bunch of steps, ornithine was re-created. As this happened, more and more ammonia was converted to urea. Thus, as long as amino acids were continuously broken down in the liver, the urea cycle would spin around and around, removing ammonia so that it did not accumulate and kill the organism. It was a milestone discovery that made Krebs world famous when he published his findings in 1932 (Figure 4).

Soon after that he was fired. Like many other academics in Germany, Krebs was dismissed from his position when the Nazis came to power in 1933, either because they were Jewish, as Krebs was, or because they opposed the Nazis. Prior to 1933, Germany was a powerhouse in all areas of science with a plethora of Nobel prizes going to Germans. That abruptly ended with the rise of Adolf Hitler.

Krebs relocated to England, along with many other academics escaping from Nazi controlled lands. Although he was unable to bring most of his personal possessions, he did take most of his lab equipment, including the Warburg manometer that had proven so useful in unlocking the secrets of the urea cycle.

The glycolysis pathway: Embden and Meyerhof

With the urea cycle behind him, Krebs wanted to focus on tracking what happened to carbohydrates in the cell. While at the University of Sheffield, Krebs set up the manometer and started working out the chemistry. One of his major goals was to map out the ultimate fate of glucose in the presence of oxygen. By this time, the initial breakdown of glucose was already understood, step-by-step. Known as glycolysis, this initial process splits each glucose molecule into two smaller molecules called pyruvate.

The steps of glycolysis were worked out by two biochemists, Gustav Embden and Otto Fritz Meyerhof. (A few years after Krebs, Meyerhof also fled Nazi Germany for being Jewish.) Unlike burning glucose to a crisp in the laboratory, the conversion of glucose to pyruvate in cells is carefully controlled by enzymes. Each step in the Embden-Meyerhof glycolysis pathway has its own enzyme that performs a specialized procedure on one molecule after another, like the factory worker at a particular workstation.

In the course of breaking down glucose into pyruvate, glycolysis provides the cell with some energy, and does not require oxygen (Figure 5). This is good, since many organisms live in environments where oxygen is not even available. In fact, today we know that the enzymes controlling glycolysis emerged extremely early in the history of life, before there was any oxygen gas in Earth’s oceans or atmosphere.

The understanding of glycolysis left a big question: What happens to the pyruvate after it is produced from the breakdown of glucose? By Krebs’ time, it was known that the answer depended on whether or not oxygen was available. It was also known that certain microorganisms, as well as animal muscles, produce a chemical compound called lactic acid. The reason, it turns out, is that lactic acid is very similar to pyruvate. When no oxygen is available – or in organisms that don't have the ability to use oxygen even if it is available – pyruvate is converted to lactic acid as a waste product. This is what happens in muscle cells during intensive exercise, especially in an individual who has not warmed up sufficiently.

However, as Krebs knew, something bigger must have been happening in cells when oxygen was available. One reason warming up helps muscles is that it brings more oxygen into the muscle cells, allowing for conversion of pyruvate to something other than lactic acid. Oxygen, it turns out, allows cells to activate a highly efficient system to break down fuel to the ultimate end product: carbon dioxide (CO2).

The Krebs cycle

When 19^th century researchers burned sugar in the lab, they knew that oxygen was required to fuel the fire. This suggested that the metabolism of glucose also required oxygen, at least when glucose was broken down all the way to CO₂ and H₂O. Krebs knew that the key to understanding how most of the energy was extracted from glucose was to understand what happened to pyruvate when oxygen was present. Clearly, it was something different than what happened in the absence of oxygen. Think of a fork in the road at the point that pyruvate is created from the breakdown of glucose. Without oxygen, pyruvate is converted to lactic acid, but the presence of oxygen opens the gate to an alternate route that ends, not with lactic acid, but with CO₂. All that Krebs needed to do was figure out the various steps that occurred along the way. Luckily, he still had his handy manometer, and luckily, he didn't need to start from scratch. A few reactions that Krebs was about to discover as steps in his new cycle were known already as independent reactions from research of an older biochemist, Albert Szent-Györgyi. It was Krebs who postulated that the reactions might be connected in a cycle, just like the reactions of the urea cycle that he'd discovered back in Germany.

Krebs’ research method was to let slices of beef liver soak in solutions of various chemicals. Using the Warburg manometer, Krebs could then see how the unidentified liver enzymes would change the different chemicals in the solutions. Testing the reactions one by one, he discovered that the breakdown of carbohydrates, lipids, and proteins did indeed proceed in a cyclic fashion. Bigger and more complex than the urea cycle, this cycle turned out to be the central route of all metabolic activity in the cell. Krebs identified the cycle’s reactions by 1937, although he tweaked it over the course of the following decade. Part of that tweaking led him to discover yet one more cycle, a little one called the glyoxylate cycle that acted as a bypass route for a section of the Krebs cycle.

Moving molecules from workstation to workstation

Thinking about the Krebs cycle in terms of workstations is a way to remember broadly what types of chemical compounds enter the cycle at certain points, what they are changed into as a result of entering the cycle, and what compounds then leave the cycle at different points. Since it is a circular pathway, there is no beginning or end. For the sake of learning the Krebs cycle, however, the “first” reaction – is the conversion of oxaloacetate into citrate. While there are several differences between oxaloacetate and citrate, the most important difference is that citrate is the bigger molecule. Its “backbone” is built of six carbon atoms, while oxaloacetate has just four. What is the source of the two extra carbon atoms? The chemical equations that Krebs wrote out told him that the source of the two carbon atoms could be acetate, which had to come from outside the cycle. Mixing oxaloacetate with his liver specimens, Krebs could test his hypothesis. Using the Warburg manometer to measure changes in oxygen and CO₂ in his mixture, Krebs could tell when the cycle was turning around. The liver specimens supplied the enzymes that controlled the reactions, including the enzyme that adds two carbons to oxaloacetate, forming citrate. This meant that Krebs could add different carbon sources, one by one, to the mixture, and see which, if any allowed the cycle to go around. Doing this, he confirmed that acetate was the needed substrate. In a test tube, as in the cells of his liver specimens, acetate had to be supplied from outside the cycle. Otherwise, the cycle would come to a halt.

The discovery that acetate joined a cycle of reactions that led to the extraction of energy from food was a major insight, because both sugars and fats – the major sources of dietary energy for all organisms – can be broken down to acetate, as can some amino acids. Krebs realized that pyruvate, the product of the initial breakdown of glucose, is very similar to acetate, except that pyruvate has one additional carbon. If the extra carbon from pyruvate were removed, the remaining molecule was easily converted to acetate.

The acetate molecule itself is small, and highly diffusible, so it must be chaperoned around the cell by a much larger carrier molecule, called co-enzyme A (Co-A). Once the two-carbon acetate is linked up with the four-carbon oxaloacetate, however, the Co-A is free to pick up another acetate and repeat the process. Meanwhile, the cell has a new molecule of citrate with its six carbon atoms.

Realizing that he was dealing with a cyclic pathway, Krebs discovered that citrate does not remain for very long. After its shape is changed around, it is cut down to a five-carbon molecule and then again to a four-carbon molecule, which then is modified several times until oxaloacetate is produced, all ready to be combined with a new acetate to produce more citrate, and the cycle goes around another time. It’s a true cycle, because the product of the cycle – oxaloacetate – is also the first ingredient for the next cycle. (See Figure 6.)

Where do the carbon atoms go when they get cut off as the cycle goes from six to five and back to four-carbon units? The cycle occurs only in aerobic organisms, life forms that use oxygen, and using the Warburg manometer Krebs discovered that a portion of the carbon removed from the main compound was combining with oxygen atoms to generate CO₂.

ATP, the cellular energy currency

The energy contained in fatty acids, glucose, and amino acids is held in the various chemical bonds that keep the individual atoms together. The most common way that we store the energy harvested from those chemical bonds is within a molecule called ATP. ATP is often called the cellular currency of energy. Just like economic currency, such as a dollar bill, the energy currency of ATP can be used to “purchase” whatever reactions or activities the cell needs to perform. Energy metabolism also depends on a chemical compound called GTP, which is nearly the same as ATP. Continuing with the dollar bill analogy, one can imagine GTP as a silver dollar coin. It’s not encountered as often as the normal paper dollar, but it has the same value and the two can be exchanged easily. As biological fuel such as glucose is broken down, ATP and GTP molecules are produced at different points in the assembly line.

Krebs found that the breakdown of sugars and fats into CO₂, through a cycle of many chemical reactions, produced ATP and GTP that the cell could use to drive all sorts of reactions and activities. However, something was clearly missing. For one thing, the amount of ATP and GTP was much smaller than he predicted. Scientists knew how many calories sugars and fats provided and most of that energy was still unaccounted for in the reactions that Krebs discovered. Secondly, oxygen was not directly required for any of the reactions that Krebs discovered. Why was oxygen so important for harvesting the energy of sugars and fats if it wasn't required in their breakdown?

Two new energy carriers

Looking more closely at chemical bonds of food molecules, the energy that is first present in those bonds is actually carried by the electrons that form the covalent bonds. Each bond is made by a pair of electrons, and depending on how the various atoms are arranged, the electron pairs can hold different amounts of energy. In the course of chemical reactions that harvest the energy from the food molecules to form ATP and GTP, the energy-holding electron pairs are physically transferred from one chemical compound to another, and various compounds that carry the electrons around are called electron carriers.

During the course of the Krebs cycle, two compounds are produced that do not feed back into the cycle. They are not ATP or GTP, and their function was not obvious to Krebs. These compounds were NADH and FADH₂, made from their precursors NAD⁺ and FADH⁺, as shown in Figure 7 (learn more about these compounds in our Energy Metabolism II module). Krebs knew that NADH was also made in glycolysis, so he suspected that finding out what they do would probably answer the question of where the rest of the ATP in cellular respiration comes from.

The discovery of the Krebs cycle would earn Hans Adolf Krebs the Nobel Prize in Physiology and Medicine in 1953, and five years later a knighthood. Even though Krebs did not discover the next phase of cellular respiration – oxidative phosphorylation - his work with the urea cycle and the Krebs cycle probably helped to inspire those discoveries, because, as it turned out, oxidative phosphorylation was also a kind of assembly line.