Structure and Functions Glycolysis is the first step in the process that cells use to extract energy from food molecules. Although energy can be extracted from most types of food molecule, glycolysis is usually considered to begin with glucose. In fact, the term “glycolysis” actually means the splitting (lysis) of glucose (glyco). This is a good description for the process, since the glucose molecule is split into two halves. The glucose molecule consists of a backbone of six carbon atoms to which are attached, in various ways, twelve hydrogen atoms and six oxygen atoms. The glucose molecule is inherently stable and unlikely to split spontaneously at any appreciable rate.
When the energy is extracted from a glucose molecule, it is stored, for the short term, in a much less stable molecule called adenosine triphosphate (ATP). The ATP molecule consists of a complex organic molecule (adenosine) to which are attached three simple phosphate groups (see figure).
ATP consists of a five-carbon sugar called ribose, linked on one side to the nitrogenous base adenine and on the other side to a linear chain of phosphate groups. The molecule formed by the attachment of adenine to ribose is called adenosine, and the linkage of three phosphates generates adenosine triphosphate. The first phosphate is attached to the ribose sugar by means of a chemical bond whose energy is no greater than those bonds found anywhere else in the molecule. While the first phosphate is attached by what one could call a “normal” chemical bond, the second and third phosphates are attached by high-energy bonds. These are chemical bonds that require a considerable amount of energy to create. Thus ATP is an ideal energy storage molecule that provides readily available energy for the biosynthetic reactions of the cell and other energy-requiring processes.
When one of the high-energy bonds of ATP is broken, a large amount of energy is released. Usually, only the bond holding the last phosphate is broken, producing a molecule of adenosine diphosphate (ADP) and a free phosphate group. The phosphate group is only split from ATP at the precise moment when energy is required by some other process in the cell. This breaking of ATP provides the energy to drive cellular processes. The processes include activities such as the synthesis of molecules, the movement of molecules, and the contraction of muscle. The third phosphate can be reattached to ADP using energy released from glycolysis, or by other components of cellular respiration. The production of ATP can be diagrammed as follows: “energy from glycolysis + ADP + phosphate → ATP.” Similarly, the breakdown of ATP can be diagrammed as “ATP → ADP + phosphate + usable energy.” With this understanding of how ATP works, one can look at how it is generated in the cell by glycolysis.
The first step in the production of energy from sugar is really an energy-consuming process. Since glucose is inherently a stable molecule, it must be activated before it will split. It is activated by attaching a phosphate group to each end of the six-carbon backbone. These phosphate groups are supplied by ATP. Therefore, glycolysis begins by using the energy from two ATP molecules. The atoms of the glucose molecule are also rearranged during the activation process so that it is changed into a very similar sugar, fructose. A fructose molecule with a phosphate group on either end is called fructose 1,6-diphosphate. Thus one can summarize the activation process as “glucose + 2 ATP → fructose 1,6-diphosphate + 2 ADP.”
Fructose 1,6-diphosphate is a much more reactive molecule and can be readily split by an enzyme called aldolase into two three-carbon compounds called dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). DHAP is converted into G3P by an enzyme called triose phosphate isomerase, which makes G3P the starting point for all the following steps of glycolysis. Each G3P undergoes several reactions, but only the more consequential reactions will be mentioned. G3P undergoes an oxidation reaction, catalyzed by an enzyme called glyceraldehyde 3-phosphate dehydrogenase. Oxidation reactions involve the loss of high-energy electrons. Electrons are highly energetic and have a negative electrical charge. They are picked up and carried by molecules specially designed for this purpose.
These energy-carrying molecules are called nicotinamide adenine dinucleotide (NAD). Biologists have agreed on a conventional notation for this molecule to allow the reader to know whether the molecule is carrying electrons or is empty. Since the empty molecule has a net positive charge, it is denoted as NAD+. When full, it holds a pair of electrons. One electron would neutralize the positive charge, while two result in a negative charge. The negative charge attracts one of the many hydrogen ions (H+) in the cell. Thus when carrying electrons the molecule is denoted NADH. G3P surrenders two high-energy electrons to NAD+. The G3P molecule also picks up a free phosphate group at the end opposite from where one is already attached to form 1,3-bisphosphoglycerate. One can summarize the reaction as “2 Glyceraldehyde 3-phosphate + 2 NAD+ + 2 inorganic phosphates → ? 2 1,3-bisphosphoglycerate + NADH + H+.” The following reactions merely transfer the energy in these chemical bonds to high-energy bonds by transferring these phosphate groups to ADP molecules to produce ATP. Since each G3P eventually produces two ATPs, and two G3Ps are produced from each original glucose molecule, glycolysis produces four ATP molecules all together. However since two ATPs were used to activate the glucose, the cell has a net gain of two ATP molecules for each glucose molecule used.
The rearrangement of the atoms leaves them in a form called pyruvate. Pyruvate still contains much energy locked up in its chemical bonds. In most of the cells of the body and most of the time, pyruvate will be further broken down and all of its energy released. This further breakdown of pyruvate requires oxygen and is beyond the scope of this topic. It should be pointed out, however, that the complete breakdown of two molecules of pyruvate can produce more than thirty additional ATP molecules. With the addition of oxygen, the end products are the simple molecules of carbon dioxide and water.
The oxidative pathways that completely break down pyruvate are limited by the lack of oxygen in very active muscles. The ability to deal with electrons from NADH is also drastically reduced. Glycolysis can continue even in the absence of oxygen, but the electrons produced by glycolysis must be dealt with.
There is a very limited amount of NAD+ in each cell. NAD+ is designed to hold electrons briefly, while they are transferred to some other system. In the absence of oxygen, the electrons are transferred to pyruvate. Since pyruvate cannot be broken down without oxygen, there is an ample supply. Transferring electrons from NADH to pyruvate allows the empty NAD+ to pick up more electrons produced by glycolysis. Therefore, glycolysis can continue producing two ATP molecules from each glucose molecule used. While two ATPs per glucose molecule is a small amount compared to the more than thirty ATPs produced by oxidative metabolism, it is better than none at all.
The process of generating energy (ATPs) in the absence of oxygen is referred to as fermentation. Most people are familiar with the fermentation of grapes to produce wine. Yeast has the enzymes to transfer electrons from NADH to a derivative of pyruvate and to convert the resulting molecule into alcohol and carbon dioxide. No further energy is obtained from this process. Alcohol still contains much of the energy that was in glucose. Humans and other mammals have different enzymes than yeast cells. These enzymes transfer the electrons from NADH to pyruvate, producing lactate.
Glycolysis and Muscle Activity When yeast is fermented anaerobically (without oxygen), it will continue producing alcohol until it poisons itself. Most yeast cannot tolerate more than about 12 percent alcohol, the concentration found in most wine. The lactate produced by fermentation in humans is also poisonous. People, however, do not respire completely anaerobically. The two ATPs produced per glucose molecule used are simply not enough to supply the energy needs of most human cells. Muscle cells have to be somewhat of an exception. There are times when one asks the muscle cells to use energy much faster than one can supply them with oxygen. One may consider a muscle working under various levels of physical activity and examine its oxygen requirements and waste products.
At rest, a muscle requires very little ATP energy. For an individual sitting on the couch watching television, energy demands are minimal. The lungs inhale and exhale slowly and take in enough oxygen to keep its concentration in the blood high. A relatively slow heart rate can pump enough of this oxygen-rich blood to the muscles to supply their very minimal needs. As soon as one uses a muscle, however, its ATP consumption increases dramatically. Even if an individual simply walks as far as the refrigerator, large quantities of ATP are required to cause the leg muscles to contract. Muscle cells maintain a constant level of ATP so that, as soon as one asks a muscle to contract, it can do so. The ATP that is broken down is almost instantly regenerated from an additional energy store peculiar to muscle cells. Creatine phosphate is a molecule similar to ATP, in that the phosphate group is attached by a high-energy bond. There is more creatine phosphate in muscle cells than ATP. As soon as ATP is broken down, phosphates, and their high-energy bonds, are transferred from
creatine phosphate. Within the first few seconds of activity, the ATP concentration in a muscle cell remains almost constant, but the creatine phosphate level begins to drop.
As soon as the creatine phosphate concentration drops, the aerobic (oxygen-requiring) respiratory processes speed up. These processes break down glucose all the way to carbon dioxide and water and release plenty of ATP. This ATP can then be used for muscle contraction. If the muscle has now stopped contracting, the new ATP produced will be used to rebuild the store of creatine phosphate.
Within the first minute or so of muscle contraction, the use of oxygen can be quite high. The circulatory system has not yet responded to this increased oxygen demand. Muscle tissue, however, has a reserve of oxygen. The red color of most mammalian muscles is attributable to the presence of myoglobin, which is similar to hemoglobin in that it has a strong affinity for oxygen. The myoglobin stores oxygen directly in the muscle, so that the muscle can operate aerobically while the circulatory and respiratory systems adjust to the increased oxygen demand.
At low or moderate muscle activity, the carbon dioxide produced by aerobic respiration in muscles will trigger an increase in the activity of both the circulatory and the respiratory systems. The increased demand for oxygen by the muscles is supplied by an increased blood flow. Jogging around a track or participating in aerobic exercises would be considered low to moderate muscular activity. Respiration rate and pulse rate both increase with jogging. This increase in oxygen supply to the muscles provides all that they need. The level of creatine phosphate will be lower than that in resting muscles, but it will soon be replenished when the activity is stopped. The muscle cells have a good supply of food molecules in the form of glycogen. Glycogen is simply a long string of glucose molecules connected together for convenient storage. At a rate of activity such as that created by jogging, the glycogen supply can last for hours. Even after it is used up, glycogen stored in the liver can be broken down to glucose and carried
to the muscles by the blood. An individual will probably want to stop jogging before his or her muscles will want to quit.
High levels of muscular activity pose a different set of problems. After more than about a minute of vigorous exercise, the muscles begin to use ATP faster than oxygen can be supplied to regenerate it. The additional ATP is supplied by lactic acid fermentation. Glucose is only broken down as far as pyruvate, then converted to lactate by the addition of electrons from NADH. Lactate begins to accumulate in the muscle tissue. Since the body is still using large amounts of ATP but not taking in enough oxygen, it is said to enter a state of oxygen debt. When the muscular activity ends, the oxygen debt is repaid.
One can use an example of someone running to catch a bus, sprinting for fifty yards at full speed. That is not enough time for the circulation and lungs to respond to the increased demand for oxygen. The muscles have made up the difference between supply and demand with lactic acid fermentation. The individual now sits down in the bus and pants—to repay his or her oxygen debt.
Some of the oxygen will go to replenish the store in muscle myoglobin. Some of it will be used in oxidative metabolism in the muscle to replenish the reserves of creatine phosphate. The rest will be used to deal with the accumulated lactate. The lactate is not all dealt with in the muscle where it was produced. Being a small molecule, it easily enters the bloodstream. In muscles throughout the body, it can be converted back to pyruvate. Pyruvate can then reenter the oxidative pathway and be used to generate ATP, with the use of oxygen. The lactate, then, is being used as a food molecule to supply the needs of resting muscle. Much of the lactate is metabolized in the liver. Some of it will be metabolized with oxygen to produce the energy to convert the rest of it back to glucose. The glucose can then be circulated in the blood or stored in the liver or muscles as glycogen. A minimal amount of lactate is excreted in the urine or in sweat.
If the subject of the preceding example kept running at full speed, having missed the bus and run all the way to the office, lactate would build up in the muscles and in the blood. If the office was far enough away, the subject would eventually reach the point of exhaustion and stop running. At that point, the level of lactate in the leg muscles would be high enough to inhibit the enzymes of glycolysis. Glycolysis would slow down so that lactate would not become any more concentrated. The muscles’ supply of creatine phosphate would be almost exhausted, but the ATP supply would be only slightly lower than in a resting muscle. The body is protected from damaging itself: Too much lactate would lower the pH to dangerous levels, and the absolute lack of ATP causes muscles to lock, as in rigor mortis. The body’s self-protection mechanisms force one to stop before either of these conditions exists. Once the subject stops running, and pants long enough, he or she can continue. The additional oxygen taken in by increased respiration will have metabolized a sufficient amount of lactate to allow the muscles to start working again.
In cases where an individual has an inherited deficiency of particular enzymes of glycolysis, the consequences for muscle tissue are rather dire. Muscles, which depend heavily on glycolysis when operating under conditions of oxygen debt, fail to perform well if any of the glycolytic enzymes are defective. Symptoms include frequent muscle cramps, easy fatigability, and evidence of heavy muscle damage after strenuous exertion.
Glycolysis and Red Blood Cell Function Red blood cells are the oxygen-ferrying units of the bloodstream and are filled with an iron-containing protein called hemoglobin. Hemoglobin binds oxygen tightly when oxygen concentrations are high and releases oxygen when oxygen concentrations are low. To perform their task successfully, red blood cells must maintain the health and functionality of their hemoglobin stores, and glycolysis helps them do that. In red blood cells, approximately 90 to 95 percent of the glucose that enters the cell is metabolized to lactate by means of glycolysis and lactate dehydrogenase. The ATP generated by glycolysis is used to bring charged atoms into the cell such as calcium, potassium, and others. The NADH generated by glycolysis is also used to maintain the iron found in hemoglobin in a state that allows it to bind oxygen. Glycolysis is also used to form the metabolite 2,3-DPG (2,3-Diphosphoglycerate). 2,3-DPG binds to hemoglobin and forces it to release oxygen more readily when oxygen concentrations are low. Thus 2,3-DPG aids hemoglobin delivery of oxygen to the tissues.
Abnormalities in the enzymes that catalyze the reactions of glycolysis are inherited. Individuals who inherit two copies of a gene that encodes a mutant form of a glycolytic enzyme experience uncontrolled destruction of red blood cells (hemolysis). The red blood cell destruction that results from defects in glycolytic enzymes is chronic and not ameliorated by drugs. An enlarged spleen is a typical symptom of glycolytic enzyme abnormalities, as the spleen tends to fill with dying red blood cells. The red blood cell destruction can be so severe that blood transfusions might be necessary. Removal of the spleen reduces red blood cell destruction.
Insulin, Diabetes, and Glycolysis Glycolysis is heavily regulated by the hormones insulin and glucagon. Insulin, a hormone made and released by the beta cells of the pancreatic islets, stimulates the insertion of the GLUT4 glucose transporter into the membranes of cells. People with type 1 diabetes mellitus, who are incapable of making sufficient quantities of insulin, tend to have very high blood sugar readings, since their cells cannot receive the signal to insert the glucose transporter into their membranes and take up glucose from the blood. This prevents the removal of glucose from the blood, and in type 1 diabetics the blood glucose level climbs to abnormally high levels. GLUT4 allows the uptake of glucose without the input of energy. Therefore, glycolysis occurs as fast as the cells can take up glucose.
Insulin also stimulates the synthesis of a metabolite called fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is a potent activator of phosphofructokinase, and activation of this enzyme ensures the activation of glycolysis. Insulin also activates the expression of genes that encode the protein involved in glycolysis. During uncontrolled diabetes, reduced glucose transport in muscle inhibits muscle cell glycolysis. In liver cells, reduced glycolytic gene expression and attenuation of the levels of fructose 2,6-bisphosphate reduce glycolysis. This contributes to the voluntary muscle weakness, liver dysfunction, and heart problems that are sometimes observed in diabetics.
Glycolysis and Cancer
The uptake of glucose and its degradation by glycolysis occurs ten times faster in tumor cells than in nontumor cells. This phenomenon, called the Warburg effect, seems to benefit tumor cells, since they lack an extensive capillary network to feed them oxygen and must rely on anaerobic glycolysis to generate ATP.
Oxygen-poor conditions also induce the synthesis of a protein called hypoxia-inducible factor (HIF). HIF is a transcription factor that helps turn on the expression of specific genes that help cells survive oxygen-poor conditions. The synthesis of at least eight glycolytic enzymes are activated by HIF. These fundamental observations of cancer cells have shown that glycolytic enzymes are excellent potential drug targets for anticancer agents.
Perspective and Prospects Cellular respiration is the process by which organisms harvest usable energy in the form of ATP molecules from food molecules. Lactic acid fermentation is the form of respiration used by human muscles when oxygen is in limited supply. Glycolysis is the energy-producing component of lactic acid fermentation, which is much less efficient than aerobic cellular respiration. Fermentation harvests only two molecules of ATP for every glucose molecule used, while aerobic respiration produces a yield of more than thirty molecules of ATP. Most forms of life will resort to fermentation only when oxygen is absent or in short supply. While higher forms of life such as humans can obtain energy by fermentation for short periods, they incur an oxygen debt that must eventually be repaid. The yield of two molecules of ATP for each glucose molecule used is simply not enough to sustain their high demand for energy.
Nevertheless, lactic acid fermentation is an important source of ATP for humans during strenuous physical exercise. Even though it is an inefficient use of glucose, it can provide enough ATP for a short burst of activity. After the activity is over, the lactate produced must be dealt with, which usually requires the use of oxygen.
Most popular exercise programs focus on aerobic activity. Aerobic exercises do not place stress on muscles to the point where the blood cannot supply enough oxygen. These exercises are designed to improve the efficiency of the oxygen delivery system so that there is less need for anaerobic metabolism. Training programs in general attempt to tune the body so that the need for lactic acid fermentation is reduced. They concentrate on improving the delivery of oxygen to the muscles, storing oxygen in the muscles, or increasing the efficiency of muscular contraction.
Insulin signaling activates glycolysis whereas another pancreatic peptide hormone, glucagon, inhibits glycolysis. Diabetics can suffer from inadequate glycolytic activity in particular organs, which can result in organ dysfunction. Expression of mutant forms of various glycolytic enzymes or supporting enzymes in transgenic mice has elucidated the link between abnormalities in glycolysis and the pathology of diabetes mellitus.
In the 1920s the German biochemist Otto Warburg demonstrated that cancer cells voraciously take up glucose and metabolize to lactate. Glycolysis is very active in cancer cells and helps them flourish under low-oxygen conditions. The development of new glycolytic inhibitors may constitute a new class of anticancer drugs that have wide-ranging therapeutic applications.
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