Course: PGPathshala-Biophysics Paper 3: THERMODYNAMICS OF LIVING SYSTEMS AND BIOENERGETICS Module 13: ENERGY GENERATION: GLYCOLYSIS Content Writer: Dr. Radhika Bakhshi, Shaheed Rajguru College of Applied Sciences, University of Delhi 1. Introduction A basic property of all living organisms is to harness energy and channel it into biological work, ie. to perform various processes to stay alive, to grow and to reproduce. These life processes require nutrient molecules taken in, to extract energy and also to provide the building blocks to create new molecules. Thus chemical energy of fuels is used to synthesize complex, highly ordered macromolecules from the simpler precursors. The organisms also convert the chemical energy into concentration gradients and electrical gradients, into motion and heat; and in some organisms (like the firefly) into light. Photosynthetic organisms convert the light energy into all these other forms of energy. The energy-generating process takes place via a series of many small steps, in which electron donors transfer energy to electron acceptors. The monocaccharide, glucose, occupies a central position in the metabolism of plants, animals and many microorganisms. The oxidation-reduction reactions are fundamental to the extraction of energy from molecules, like glucose. The principal electron carriers are NADH and its oxidized form, NAD +. The ultimate electron acceptor in the complete oxidation of glucose is an oxygen atom, which forms water. Energy generated in this reaction is conserved by the conversion of low energy ADP to high energy ATP. This ADP- ATP system is like an active savings bank account, in which deposits and withdrawals are in a steady state. Energy from ATP is never used up, just transformed from one form to the other for the various energy-requiring processes of the cell. Glycolysis is the first in the series of pathways for the oxidation of glucose. The subject content on Energy Generation and Glycolysis has been discussed in detail spreading over 2 modules. The major Objectives of these Modules are given below: 2. Learning Outcome General introduction of metabolism and the types of metabolic pathways. Metabolic fuels used by organisms The central theme of metabolism Glucose utilization Glycolysis details of the pathway and its importance 3. Metabolism All living organisms need a continuous influx of free energy. This energy is required to maintain order in a universe bent on maximizing disorder. Metabolism is the overall process through which living systems acquire and utilize free energy they need to carry out their various functions. This is done by coupling the exergonic reactions (energy-releasing) of nutrient oxidation to the endergonic processes (energy requiring). This is required to maintain to carry out the various biological functions like, active transport of molecules against concentration gradients, biosynthesis of complex molecules and perform mechanical work. The free energy is most often coupled to endergonic reactions through the intermediate synthesis of high-energy phosphate compounds such as ATP (adenosine triphosphate). In addition to being completely oxidized, nutrients are broken down in a series of metabolic reactions to common intermediates. These intermediates are then used as precursors in the synthesis of other biological molecules. The organisms are classified on the basis of the mode in which they acquire the free energy: 1
1. Phototrophs- acquire free energy from the sun, through the process in which light energy powers the endergonic reaction of CO 2 and H 2 O to form carbohydrates. This is called as photosynthesis. Plants and certain bacteria are examples. 2. Chemotrophs- obtain their free energy by oxidizing organic compounds (like carbohydrates, lipids and proteins) obtained from other organisms, ultimately phototrophs. Animals and nonphotosynthetic bacteria are examples. A 40-year old normal human adult consumes tons of nutrients and around 20,000 L of water, but without significant change in weight. This steady state is remarkably maintained by a sophisticated set of metabolic regulatory systems. Metabolic Pathways are series of consecutive enzymatic reactions that produce specific products. Metabolites are the reactants, intermediates and products of such reactions. Metabolism comprises of two types of reaction pathways: 1. Catabolic Pathways (degradative) - in these pathways, the nutrients and cell constituents are broken down exergonically to salvage their components and/or to generate free energy. 2. Anabolic Pathways (biosynthetic) in these pathways, biomolecules are synthesized from simpler components. Catabolic processes release free energy, which is conserved through the synthesis of ATP from ADP (Adenosine diphosphate) and phosphate or through the reduction of the coenzyme NADP+ to NADPH. ATP and NADPH are the major free energy sources for anabolic pathways. 3.1. Metabolic fuels are substances that are used by the body as sources of carbon or oxidized to release free energy, which is used to support anabolic processes and other cellular functions. The four kinds of molecule, used as major metabolic fuels are: Carbohydrates Fatty Acids Ketone bodies Amino Acids Carbohydrates are stored primarily as glycogen in the liver and skeletal muscle and to some extent in other tissues. Circulating glucose in the blood is a major metabolic fuel and a number of mechanisms are used to maintain the appropriate blood glucose level. Caloric Value of metabolic fuels the heat evolved when an organic substance is reacted with molecular oxygen in a bomb calorimeter and all carbon converted to CO 2. It is a measure of the potential free energy available in the substance and expressed in kilocalories (kcal) per gram. Metabolic Fuel Caloric Value (kcal/g) Carbohydrates (glucose) 4 Amino Acids (average) 4 Ketone bodies (hydroxybutyrate, acetoacetate) 4 Fatty acids 9 3.2. Central theme of metabolism A striking feature of catabolism is that it converts the metabolic fuels to common intermediates (like acetyl-coenzyme A). These are then further metabolized in a central oxidative pathway, that terminates in a few end products. 2
Figure 1. Central Theme of Metabolism 3 From Lehninger Principles of Biochemistry, Fifth
Biosynthetic carries out the opposite processes. Relatively few metabolites, mainly pyruvate, acetyl- CoA and the citric acid cycle intermediates, serve as starting materials for a host of different biosynthetic products. 3.3. Principal Characteristics of metabolic pathways: 1. These pathways are irreversible. 2. Catabolic and Anabolic pathways always differ for a metabolite. 3. Every metabolic pathway has a first committed step 4. All pathways are regulated. 5. Metabolic pathways in eukaryotic cells occur in specific cellular locations. 4. Glycolysis It is first stage of glucose metabolism and was the first biochemical pathway to be elucidated. It is an anaerobic process that, by itself yields only 2 molecules of ATP. The complete aerobic oxidation of glucose to CO 2 and water (involving glycolysis, the citric acid cycle and oxidative phosphorylation) yields the energy equivalent of 32 molecules of ATP. The following are some of the fates of glucose. Although these are not the only possible fates for glucose, these are the most significant. Figure 2. Major pathways of glucose utilization. From Lehninger Principles of Biochemistry, Fifth Edition Fates of pyruvate: Under aerobic conditions, the pyruvate formed by glycolysis is further oxidized by the citric acid cycle and oxidative phosphorylation to CO 2 and water. Under anaerobic conditions, the pyruvate is converted to a reduced end product, by the process of fermentation: Lactic Acid Fermentaion Pyruvate converted to Lactate in muscle. Alcoholic Fermentation Pyruvate converted to ethanol and CO 2 in yeast. 4
4.1. Importance of Glycolysis It is the pathway by which glucose is converted to pyruvate (via intermediate fructose-1,6- bisphosphate), with the generation of 2 molecules of ATP per molecule of glucose. It is first metabolic pathway in the catabolism of glucose. It plays a key role in energy metabolism by providing a significant portion of the energy utilized by most organisms It prepares glucose and other carbohydrates for oxidative degradation. In sudden bursts of energy, carbohydrates are used faster by the body, as compared to them being utilized aerobically. The pyruvate thus formed is converted to lactate, which is eventually exported from the muscle to the liver. Under aerobic conditions, the main purpose of glycolysis is to feed pyruvate into the citric acid cycle, where further metabolic steps will yield much more ATP. Figure 3. Possible fates of pyruvate Source-Pearson education 5
4.2. Overview of Glycolysis Source of glucose in the blood is usually from the breakdown of higher polysaccharides or from its synthesis from non-carbohydrate sources (gluconeogenesis). It enters most cells by a specific carrier (glucose transporter) that transports it from the exterior of the cell into the cytosol. Glycolytic enzymes are located in the cytosol. In glycolysis, one molecule of glucose (a six-carbon compound) is converted to fructose-1,6- bisphosphate (also a six-carbon compound), which eventually gives rise to two molecules of pyruvate (a three carbon compound). In essence, the glycolytic process converts glucose to two pyruvate molecules of lower free energy, in a process that harnesses the released free energy to synthesize ATP from ADP and inorganic phosphate (Pi). The 10 enzyme-catalyzed reactions of glycolysis are divided into two stages: Phase I (Reactions 1-5)- Preparatory phase. This process utilizes two ATPs as an energy investment. Phase II (Reactions 6-10)- Payoff phase, this phase generates of four ATPs. Since stage I consumes 2 ATPs and stage II generates 4 ATPs, there is a net generation of 2 ATP molecules. The overall reaction is: Glucose + 2NAD + + 2ADP +2Pi 2NADH + 2Pyruvate + 2ATP + 2H 2 O + 4H+ 4.3. Various fates of Pyruvate - the end product of Glycolysis The pyruvate formed during the process of glycolysis can have one of the several fates. Aerobic metabolism- in the presence of oxygen, pyruvate is converted to acetyl CoA, which then enters the citric acid cycle. Anaerobic metabolism- in the absence of oxygen, pyruvate can have two fatesa. Alcoholic Fermentation - pyruvate converts to acetaldehyde, which in turn, is reduced to produce ethanol. b. Lactic Acid Fermentation - pyruvate is reduced to lactate. This is common in humans. This is also called as anaerobic glycolysis, to differentiate it from conversion of glucose to pyruvate (which is simply called glycolysis). Anaerobic metabolism is the only energy source in mammalian red blood cells (since they lack mitochondria), as well as in several species of bacteria, such as Lactobacillus in sour milk and Clostridium botulinum in tainted canned foods. 4.4. Fate of NADH NAD + is the primary oxidizing agent of glycolysis. The NADH produced in the pathway must be continually reoxidized to keep the pathway supplied with NAD +, which can occur in any one of the following three common ways- 1. Lactic Acid Fermentation: Under anaerobic conditions, NAD + is regenerated when NADH reduces to lactate. This occurs in the muscle. 6
Figure 4. Lactic Acid Fermentation 2. Alcoholic fermentation- Under anaerobic conditions, pyruvate is decarboxylated to yield CO 2 and acetaldehyde, which is further reduced by NADH to yield NAD + and ethanol. This occurs in yeast. 7
Figure 5. Alcoholic fermentation 3. Under aerobic conditions, the mitochondrial oxidation of each NADH to NAD + yields 3 ATPs. 8
Figure 6. Schematic figure of Glycolysis Source- Eberly College of Science, Penn State 9
Figure 7. Details of Glycolytic Reactions: Source- en.wikibooks 10
4.5. Preparatory phase of glycolysis This phase requires two molecules of ATP and the hexose chain of glucose is cleaved into two triose phosphates. 4.5.1. Phosphorylation of Glucose Reaction 1: Conversion of Glucose to Glucose 6- phosphate This first step of glycolysis is an irreversible reaction, under intracellular conditions and catalyzed by the enzyme Hexokinase. Kinases are those enzymes, which catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor molecule. This is the first of the two priming reactions, which requires ATP. Isozymes of hexokinases- hexokinases are present in nearly all organisms, and human genome encodes for four different hexokinases (I to IV), all of which catalyze the same reaction. The one present in hepatocytes is hexokinase IV (also called glucokinase) and differs from the other forms in kinetic and regulatory properties. Hexokinases I III are present in the muscle and differ from the glucokinase in various respects. Firstly, the glucose concentration at which glucokinase is half-saturated (about 10mM) is higher than the usual concentration of glucose in blood (ie. has a higher Km than the other kinases). An efficient glucose transporter in the hepatocytes (GLUT2) rapidly equilibrates the glucose concentration in cytosol and blood. After a meal rich in carbohydrates, the blood glucose level is high, the excess glucose is transported into hepatocytes, where glucokinase converts it to glucose 6-phosphate. Since the glucokinase is not saturated at 10mM glucose, its activity continues to increase as the glucose concentration rises to 10mM or more. When the concentration of glucose is low in blood, the glucose concentration in a hepatocyte is low relative to the Km of glucokinase and the glucose generated by gluconeogenesis leaves the cell before being trapped by phosphorylation. Secondly, glucokinase is not inhibited by glucose 6-phosphate, and it continues to function when the accumulation of glucose 6-phosphate completely inhibits the other hexokinases. 4.5.2. Reaction 2- Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate The enzyme phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerisation of glucose 6-phosphate, an aldose to fructose 6-phosphate, a ketose. 4.5.3. Reaction 3- Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate In this second priming reaction of glycolysis, enzyme phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate. So, two ATP molecules have been used so far, for each molecule of glucose. 4.5.4. Reaction 4- Cleavage of Fructose 1,6 Bisphosphate This reaction is catalyzed by the enzyme fructose 1,6- bisphosphate aldolase (also called aldolase). Fructose 1,6 Bisphosphate is cleaved to yield two different triose phosphates, Glyceraldehyde 3- phosphate and Dihydroxyacetone phosphate. 4.5.5. Reaction 5- Interconversion of the Triose phosphates Of the two triose phosphates formed, only one, ie. Glyceraldehyde 3- phosphate can be directly degraded in the subsequent steps of glycolysis. The other product, Dihydroxyacetone 11
phosphate, is rapidly and reversibly converted to glyceraldehyde 3- phosphate by the enzyme, triose phosphate isomerase. This is the last reaction of the preparatory phase of glycolysis. At C-1 and C-6, the hexose molecule is phosphorylated during this phase and then cleaved to form two molecules of glyceraldehyde 3- phosphate. 4.6. Payoff phase of glycolysis This phase consists of five reactions, including the two energy-conserving phosphorylation steps. During these steps, some of the chemical energy of the glucose molecule is conserved in the form of ATP and NADH, therefore this phase is appropriately known as the payoff phase. 4.6.1. Reaction 6- Oxidation of Glyceraldehyde 3- phosphate to 1,3- Bisphosphoglycerate This is first of the two energy-conserving reactions of glycolysis that eventually leads to the formation of ATP. This reaction is catalyzed by the enzyme glyceraldehyde 3- phosphate dehydrogenase. This is the first glycolytic reaction that generates a product of high phosphoryl group transfer potential. The amount of NAD + in a cell is limiting and much less than the amount of glucose metabolized in a few minutes. If the NADH formed in this step is not reoxidized and recycled, the glycolysis would come to a halt. The recycling of NAD + is discussed in Section 4.4 4.6.2. Reaction 7- Phosphoryl Transfer from 1,3- Bisphosphoglycerate to ADP The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3- Bisphosphoglycerate to ADP, forming ATP and 3- Phosphoglycerate.The enzyme is named for the reverse reaction, in which a phosphoryl group is transferred from ATP to 3- phosphoglycerate. This formation of ATP by phosphoryl group transfer from a substrate (1,3-bisphosphoglycerate) is referred to as substrate-level phosphorylation, which is different from the respiration-linked phosphorylation (which occurs in mitochondria of cells). 4.6.3. Reaction 8 Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate The enzyme phosphoglycerate mutase catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate, using Mg+. 4.6.4. Reaction 9- Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate (PEP) The enzyme enolase catalyzes this second glycolytic reaction that generates a compound with high phosphoryl group transfer potential. It promotes the reversible removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP). PEP is a high energy compound, as it has a high standard free energy of hydrolysis ( = -61.9kJ/mol). 4.6.5. Reaction 10- Transfer of the Phosphoryl Group from PEP to ADP The enzyme pyruvate kinase catalyzes this last step of glycolysis, in which the high-energy phosphoryl group of PEP is transferred to ADP, in the presence of K + and Mg + (or Mn 2+ ). This is also an example of substrate-level phosphorylation. About half of the energy released by PEP hydrolysis ( = -61.9kJ/mol) is conserved in the formation of the phosphoanhydride bond of ATP ( = -30.5 kj/mol), and the rest (-31.4 kj/mol) constitutes a large driving force pushing the reaction toward ATP synthesis. 12
4.7. Energetics of Glycolysis The conversion of two molecules of glyceraldehyde 3-phosphate to two molecules of pyruvate is accompanied by the formation of four molecules of ATP from ADP. The net yield of ATP per molecule of glucose, however, is only two, since two molecules of ATP were invested in the preparatory phase of glycolysis to phosphorylate the two ends of the hexose molecule. The overall reaction for glycolysis under aerobic conditions is: Glucose + 2NAD + + 4ADP + 2Pi 2 Pyruvate + 2NADH + 2H+ + 2ATP +2H 2 O The two molecules of NADH formed by glycolysis in the cytosol are, under aerobic conditions, reoxidized to NAD + by transfer of their electrons to the electron-transfer chain (located in the mitochondria). The electron-transfer chain passes these electrons to Oxygen (the ultimate electron acceptor): 2NADH + 2H + + O 2 2NAD + + 2H 2 O The transfer of electrons from NADH to O 2 in mitochondria provides the energy for synthesis of ATP by respiration-linked phosphorylation. Under anaerobic conditions, the NAD + is regenerated by the conversion of pyruvate to either ethanol (as in yeast) or lactate (as in most organisms, including humans) as discussed previously. 4.8. Pasteur Effect While Louis Pasteur was studying fermentation of glucose by yeast, he observed that the rate and total amount of glucose consumption were many times greater under anaerobic than aerobic conditions. Muscle studies done later, showed the same large difference in the rates of anaerobic and aerobic glycolysis. Biochemical basis of Pasteur Effect - The ATP yield from glycolysis under anaerobic conditions yields only 2 molecules of ATP per molecule of glucose, which is much smaller than from the complete oxidation of glucose to CO 2, under aerobic conditions (30 or 32 ATP per glucose). Therefore about 15 times more glucose is consumed anaerobically, (as compared aerobically) to yield the same amount of ATP. 4.9. Regulation of Glycolysis The entry of glucose through the glycolytic pathway is regulated to maintain nearly constant levels of ATP and the glycolytic intermediates (which serve as biosynthetic precursors). This is achieved by the by a complex interplay of ATP consumption, NADH regeneration, fluctuations in the concentration of key metabolites and regulation of enzymes (namely, hexokinase, PFK-1, and pyruvate kinase). It is also regulated, in a longer time scale, by hormones glucagon, epinephrine and insulin and by the changes in expression of genes of several glycolytic enzymes. 4.10 Glycolysis and Medicine Even though the process of glycolysis is tightly regulated, it s abnormally regulated in cancer. It was observed by Otto Warburg in 1928 that many tumours have a higher rate of glycolysis, than the normal tissue, even when oxygen is available. The uptake of glucose and glycolysis proceed about 10 times faster than in normal, non-cancerous tissues. Initially the tumour cells grow under hypoxic conditions, with limited oxygen supply. Therefore, these cells must depend only on glycolysis for most of their energy needs. This yields them lower energy (only 2 ATP per glucose molecule). Therefore tumour cells must consume much more glucose than normal cells to meet their energy needs. The pyruvate generated during this process is 13
converted to lactate to regenerate NAD +. Generally, more aggressive the tumour, greater is its rate of glycolysis. This effect is made use of diagnostically. Cancerous tissue can be detected by a sensitive technique, called the Positron Emission Tomography (PET). In PET, individuals are injected with a harmless, isotopically labelled glucose analog, 2-fluoro-2- deoxyglucose (FdG). This compound in which, the C-2 of glucose is replaced with F-18, is taken up but not metabolized by the tissues. It therefore accumulates as 6-phospho-FdG, the extent of which, depends on its rate of uptake and phosphorylation by cellular hexokinase. The uptake is around 10 times higher in tumours than in normal tissues. The decay of F-18 yields two positrons per F-18 atom, which can be detected by a series of sensitive detectors positioned around the body. This allows the localization of the decaying compound and hence the tumour. SUMMARY 1 Metabolism is the overall process through which living systems acquire and utilize free energy they need to carry out their various functions. 2 a) Catabolic Pathways (degradative) - in these pathways, the nutrients and cell constituents are broken down exergonically to salvage their components and/or to generate free energy. b) Anabolic Pathways (biosynthetic) in these pathways, biomolecules are synthesized from simpler components. Catabolic processes release free energy, which is conserved through the synthesis of ATP from ADP (Adenosine diphosphate) and phosphate or through the reduction of the coenzyme NADP+ to NADPH. ATP and NADPH are the major free energy sources for anabolic pathways. 3. Glycolysis is the first stage of glucose metabolism. In this, one molecule of glucose generates two molecules of pyruvate and also 2 molecules of ATP. 4. Under aerobic conditions, the pyruvate formed by glycolysis is further oxidized by the citric acid cycle and oxidative phosphorylation to CO 2 and water. Under anaerobic conditions, the pyruvate is converted to a reduced end- product, by the fermentation: Lactic Acid Fermentaion Pyruvate converted to lactate in muscle. Alcoholic Fermentation Pyruvate converted to ethanol and CO 2 in yeast. 5. The rate of glycolysis is much greater in tumour cells, as compared to normal cells. This is used to detect cancer, by PET Scan. ----------------------------------------------------------------------------------------------------- End of Module 13 From Dr. Radhika Bakhshi Shaheed Rajguru College of Applied Sciences, University of Delhi 14