: Biochemistry of macromolecules and metabolic pathways

Transcription

1 1. 7 Metabolic pathways Metabolic pathways are chains of chemical reactions that occur in cells. Each pathway modifies chemicals using enzymes to catalyse the reactions. This topic guide looks at a metabolic pathway vital to all humans, the production of ATP by respiration. There are four distinct stages to aerobic respiration explained in this topic guide. Furthermore, the result of what happens when oxygen is not present to oxidise pyruvate at the end of glycolysis, known as anaerobic respiration, will be looked at. n successful completion of this topic you will: understand the features of and links between the major metabolic pathways (L3). To achieve a Pass in this unit you need to show that you can: summarise the function of the main metabolic pathways and the relationships between them (3.1) review the processes that control metabolic pathways (3.2) explain the apparent irreversible steps in glycolysis and gluconeogenesis exhibited by phosphofructokinase (PFK1) (3.4). 1

2 1 The stages of respiration Before you start If you find some parts of this unit challenging, remember you are working at a higher level than you may be used to. In this unit it is important that you fully understand the following themes and topics before you begin: structure and function of biological molecules enzyme structure and function aerobic respiration. If you need to check your understanding of proteins, carbohydrates, lipids and nucleic acids, Unit 2 Module 1 of CR AS Biology (P. Kennedy and F. Sochacki, 2008), offers a good introduction to the topic. If you need to check your understanding of aerobic respiration and the stages of glycolysis, link reaction, the Krebs cycle and the electron transport chain, you may find Unit 1 Module 4 of CR A2 Biology (S. Hocking, 2008) useful. Key terms Coenzymes: Work closely with enzymes to enable them to carry out their functions. They are not proteins. NAD (nicotinamide adenine dinucleotide): A coenzyme found in all living cells that carries electrons from electron donors to the electron transport chain. Glycolysis Glycolysis is the first stage of respiration. It is a two-stage process that is catalysed by enzymes and converts a molecule of glucose into two molecules of pyruvate (see Figure 1.7.1). Glucose is a six-carbon compound that is broken down into pyruvate (a threecarbon compound) using two molecules of ATP. Four molecules of ATP are produced by the process, so there is a net gain of two ATP. This stage doesn t require any oxygen and can therefore occur quickly, providing immediate energy. Glycolysis takes place inside the cytoplasm of cells. ne ATP is hydrolysed and releases a phosphate group that attaches to the glucose at carbon 6. This becomes glucose 6-phosphate, which is then changed to fructose 6-phosphate. Another ATP molecule is hydrolysed and the phosphate group is released and attaches to fructose 6-phosphate s carbon 1. This is now called fructose 1,6-bisphosphate. Both of these reactions (two phosphate groups attached to carbon C1 and C6 atoms) are catalysed by phosphofructokinase 1 (PFK1). Phosphofructokinase 1 (PFK1) is one of the most important regulatory enyzmes of glycolysis. The energy released from hydrolysing the two ATP molecules activates the hexose (six-carbon) sugar and prevents it moving out of the cell so we call this activated, phosphorylated sugar hexose 1,6-bisphosphate. Each molecule of hexose 1,6-bisphosphate is split into two molecules of triose phosphate. These are three-carbon molecules with a phosphate attached. Triose phosphate is oxidised and two hydrogen molecules with their electrons are removed from triose phosphate. This requires a dehydrogenase enzyme to remove the hydrogen, but we know it also needs a coenzyme known as NAD to help it remove the electrons. This NAD then becomes reduced NAD because it gains electrons. At this stage, two molecules of ATP are formed. Finally, two molecules of pyruvate are produced, which are three-carbon compounds. At this stage, another two molecules of ATP are formed. 2

3 Key terms NADH: Created when NAD receives electrons and is therefore reduced. This is an important molecule for respiration. Phosphorylation: The addition of a phosphate to an organic molecule. ther products are produced during this reaction overall two molecules of ATP are created along with two molecules of NADH and two molecules of pyruvate per glucose molecule. To summarise the stage of glycolysis, one molecule of glucose is broken down into two molecules of pyruvate. Phosphorylation of glucose occurs, where phosphate molecules are added to the carbons on the glucose to make fructose 1,6-bisphosphate. This is then split into two, three-carbon compounds called triose phosphate. xidation of the triose phosphate molecules occurs with NAD as the electron acceptor and these are then converted into pyruvate. The pyruvate is used to start the link reaction. The link reaction If oxygen is present, the pyruvate produced from glycolysis can be oxidised to acetate. The link reaction is the second stage of respiration and it occurs in the matrix of the organelles called mitochondria. Figure shows the basic structure of the mitochondria. Figure 1.7.1: Basic structure of a mitochondrion. Inner membrane Matrix uter membrane C C C + CoA C 2 C Key terms CoA C C NAD + NAD Figure 1.7.2: The link reaction. Acetyl coenzyme A: Needed to carry the carbon atoms within the acetyl group to the Krebs cycle, the next stage of aerobic respiration. Coenzyme A: A specific coenzyme for acetate. Decarboxylated: Carbon dioxide is removed. Dehydrogenated: Hydrogen molecules are removed. FAD (flavine adenine dinucleotide): Coenzyme similar to NAD that carries electrons from electron donors to the electron transport chain. Pyruvate dehydrogenase removes hydrogen atoms from pyruvate and pyruvate decarboxylase removes a carboxyl group from pyruvate. The coenzyme NAD accepts the hydrogen atoms and coenzyme A accepts acetate, producing acetyl coenzyme A. During the link reaction two molecules of pyruvate are processed, producing two carbon dioxide molecules, two molecules of reduced NAD and two molecules of acetyl coenzyme A (see Figure 1.7.2). There are no ATP molecules made during this stage. Krebs cycle The Krebs cycle is summarised in Figure This reaction is the third stage of respiration and it occurs in the matrix of the mitochondria. Acetate (a twocarbon compound from the link reaction) joins with oxaloacetate (a fourcarbon compound) to create citrate (a six-carbon compound). Citrate is then decarboxylated and dehydrogenated, producing a five-carbon compound, carbon dioxide and reduced NAD (NAD has gained electrons and is therefore reduced). This new five-carbon compound is also decarboxylated and dehydrogenated, producing a four-carbon compound, succinate, another carbon dioxide (C 2 ) molecule and reduced NAD. Succinate is transformed to fumarate (a four-carbon compound) and a molecule of ATP is produced. Fumarate is transformed to malate (a four-carbon compound) where FAD (flavin adenine dinucleotide) is reduced by accepting a pair of hydrogen atoms. 3

4 Finally malate is dehydrogenated to produce oxaloacetate, producing another reduced NAD molecule. For every molecule of glucose the Krebs cycle must turn twice, so the overall products of the Krebs cycle are six molecules of reduced NAD, two molecules of reduced FAD, four molecules of carbon dioxide and two molecules of ATP. Figure 1.7.3: A diagram to show the Krebs cycle. Pyruvate (3C) NAD 2H C 2 Link reaction Acetyl CoA (2C) CoA Acetate (2C) NAD FAD xaloacetate (4C) 2H 4C compound 4C compound Citrate (6C) 2H Krebs cycle C 2 ATP 4C compound 5C compound NAD 2H NAD C 2 To summarise the Krebs cycle, acetate from the link reaction joins with oxaloacetate, which produces a six-carbon compound (citrate). Carbon dioxide and two hydrogen atoms are then removed from citrate to produce a five-carbon compound. Carbon dioxide and two hydrogen atoms are removed from this five-carbon compound to produce a four-carbon compound. This four-carbon compound is converted into another four-carbon compound, during which time ATP is produced. This fourcarbon compound loses two hydrogens and forms another four-carbon compound, which also loses two hydrogens to reproduce oxaloacetate. xaloacetate is now ready to accept acetate from the link reaction again. The electron transport chain The electron transport chain is the final stage of respiration and this stage produces the most molecules of ATP. This stage consists of proteins in the membrane known as carrier molecules that act as electron acceptors. When accepting an electron, the carrier protein molecule becomes reduced. When it loses the electron to the next carrier molecule it becomes oxidised. NAD and reduced FAD produced during glycolysis, the link reaction and the Krebs cycle provide the electrons. As an electron moves along the electron transport chain (and hops from each carrier to the next) it loses energy. This energy actively transports hydrogen ions 4

5 across the inner mitochondrial membrane (see Figure on page 3), so this gives a low concentration of hydrogen ions in the inner mitochondrial membrane and a high concentration of hydrogen in the outer mitochondrial space. As this concentration gradient is generated the hydrogen ions naturally want to diffuse back to the inner mitochondrial membrane. The hydrogen ions diffuse through the membrane using ATP synthase enzymes in the membrane. As the hydrogen ions flow down the concentration gradient from a high concentration to a low concentration, ADP and Pi join to form an ATP molecule (oxidative phosphorylation). Electrons are passed from the last electron carrier to the final electron acceptor, which is oxygen. xygen binds with the hydrogen ions that have travelled through the ATP synthase, making a molecule of water. Figure is a diagram of the electron transport chain. To be able to estimate the total number of ATP molecules produced during oxidative phosphorylation we need to calculate the number of electrons provided by reduced NAD and FAD. Ten molecules per glucose of reduced NAD are produced during the first three stages and we get three ATP molecules from each of them. So 30 molecules of ATP are produced from reduced NAD. Two reduced FAD are produced in the Krebs cycle and we get two molecules of ATP from each of them, so this produces four molecules of ATP. Therefore the total ATP molecules produced during oxidative phosphorylation is 34. There were also molecules of ATP produced in previous stages two ATP produced during glycolysis and two during the Krebs cycle. In total this is 38 ATP molecules per glucose molecule. However, the two reduced NAD produced during glycolysis can be deducted as they are unable to provide the electrons to the electron transport chain, because the mitochondrion is impermeable to reduced NAD. We therefore estimate that, under the best conditions, 36 molecules of ATP are produced per molecule of glucose. Figure 1.7.4: The electron transport chain. Cytosol uter membrane Intermembrane space Complex I ox. red. UQ UQH 2 red. Complex II ox. ox. cyt. c (Fe +3 ) cyt. c (Fe +2 ) red. Complex III ox. Complex IV red. Inner membrane ATP synthase NADH NAD + FADH 2 FAD + Mitochondrium 1/ H 2 ADP ATP 5

6 Activity Answer the following questions: 1 What is the final stage of aerobic respiration? 2 Why is this stage important? 3 What happens to the electron carriers in the chain? 4 What happens to the electrons as they move along the chain? 5 Why is energy needed in the electron transport chain? 6 Where does this final stage of aerobic respiration take place? 7 Why is the production of a concentration gradient important? 8 Why are NAD and FAD important at this stage? 9 What is the final electron acceptor? 10 How many molecules of ATP are synthesised per one molecule of glucose? Gluconeogenesis Gluconeogenesis is a metabolic pathway used to generate glucose from noncarbohydrate carbon substrates such as pyruvate and lactate. This is the body s way of conserving all useful material to provide energy. Gluconeogenesis consists of a series of enzyme-catalysed reactions. Gluconeogenesis takes place mainly in the liver. Many of the reactions are the reversible steps found in glycolysis: 1 In the mitochondria, carbon dioxide is added to pyruvate to produce oxaloacetate. In this reaction one molecule of ATP is catalysed by pyruvate carboxylase. 2 xaloacetate is reduced to malate with the help of NADH, so that it can be transported out of the mitochondria. 3 Malate is then reoxidised to oxaloacetate using NAD + in the cytosol, where the following steps take place. 4 The enzyme phosphoenolpyruvate carboxykinase removes carbon dioxide and adds a phosphate group (phosphorylates) to oxaloacetate to form phosphoenolpyruvate. A molecule of guanosine-5 -triphosphate (GTP) is hydrolysed to guanosine diphosphate (GDP). 5 The enzyme fructose-1,6-bisphosphatase converts fructose-1,6-bisphosphate to fructose 6-phosphate, using one water molecule and releasing one phosphate that was added from a molecule of ATP during glycolysis. Fructose 6-phosphate then reacts with the enzyme phosphoglucoisomerase to form glucose-6-phosphate. 6 Glucose-6-phosphate is dephosphorylated to free glucose by removing the phosphate group attached to carbon 6 that was added during glycolysis. To summarise, pyruvate (from glycolysis) can be converted back to glucose by a series of enzyme-controlled reactions whereby fructose-1,6-bisphosphate produced during glycolysis is turned back to glucose by hydrolysis reactions that remove the phosphate groups that were attached to carbons 1 and 6 during glycolysis. Checklist In this topic you should now be familiar with the following ideas about metabolic pathways: respiration is necessary to all living organisms respiration of glucose occurs in four stages: glycolysis, the link reaction, the Krebs cycle and the electron transport chain ATP is the desired product of respiration it is estimated that 36 molecules of ATP are produced per glucose coenzymes play vital roles in the stages of respiration. 6

7 Activity Complete the table to show the number of molecules produced per glucose for each stage of respiration. Glycolysis The link reaction The Krebs cycle The electron transport chain C 2 ATP NAD FAD 2 Fermentation The final electron acceptor during the electron transport chain is oxygen. However, if oxygen is absent the link reaction, the Krebs cycle and the electron transport chain stop. The only stage that can still proceed anaerobically to produce ATP is glycolysis. The re-oxidisation of the reduced NAD produced enables glycolysis to continue, increasing an organism s chance of survival. Lactate fermentation Lactate fermentation happens during strenuous exercise in muscle tissue, when the demand for ATP is high but there is no oxygen. The reduced NAD produced during glycolysis needs to be re-oxidised to NAD +. Pyruvate accepts the hydrogen atoms from reduced NAD, leaving NAD available to accept more hydrogen atoms from glucose. Glycolysis is able to continue to produce enough ATP for the muscle tissue. Lactate is produced and carried to the liver. When oxygen becomes available the lactate is converted back to pyruvate and the link reaction can continue. Take it further Alcoholic fermentation occurs in yeast cells under anaerobic conditions. Visit for an overview of this process. Note down the differences that occur between lactate fermentation and alcoholic fermentation. 3 Fatty acid ß-oxidation When the body is in short supply of carbohydrates it can use its lipid supply. Triglycerides stored can be broken down into glycerol and fatty acids. ß-oxidation is a process that breaks down the fatty acids further to produce two-carbon acetyl compounds, which are then combined with coenzyme A to enter the Krebs cycle and on to the electron transport chain. The glycerol molecule is not wasted; this is converted to dihydroxyacetone phosphate and can be used in glycolysis. 7

8 Figure 1.7.5: Fatty acid ß-oxidation. Take it further There are several suitable books or websites that go into detail about the steps involved in fatty acid ß-oxidation, for instance, Biochemistry (Campbell, M.K. and Farrell, S.., 2011), Cengage Learning. This book can also be accessed online using Google books. R CH 2 CH 2 CH 2 C SCoA FAD + Dehydrogenation R CH 2 CH CH C SCoA H 2 Hydration H R CH 2 C CH 2 C SCoA H NAD + xidation R CH 2 C CH 2 C SCoA SCoA Thiolysis R CH 2 C SCoA CH 2 C SCoA Key terms adenosine triphosphate (ATP): A compound consisting of an adenosine molecule bonded to three phosphate groups, present in all living tissue and used in cells as a coenzyme. Equilibrium: Rate of both forward and backward reactions is the same. 4 Adenosine triphosphate (ATP) Adenosine triphosphate (ATP) consists of the base adenine, a ribose sugar and three inorganic phosphate groups. It is known as a high-energy molecule. When ATP is hydrolysed a large amount of energy can be released it is said to have a high free energy of hydrolysis. This free energy can then be used to drive reactions that require energy. ATP loses the terminal phosphate group during hydrolysis and becomes adenosine diphosphate; this reaction is described as exergonic. Most reactions inside cells are described as endergonic because they require energy. Free energy The body has an energy store; however, not all of this energy is available to carry out work. The amount of energy that can be used for work is known as free energy or Gibbs energy (DG). Free energy indicates how much energy is actually available for the body to carry out chemical reactions. Reactions are classified depending on their free energy changes: exergonic reactions occur with a net loss of free energy endergonic reactions need energy that proceed with a net gain of free energy endergonic reactions are coupled with exergonic reactions where ATP is necessary. Equilibrium A system can do no work at equilibrium (DG = 0). This means the rates of the forward and backward reactions are equal, and there is no change in the concentration of products or reactants. Metabolic disequilibrium is important because it means that cells are able to work throughout their life because of the constant flow of materials into and out of the cell and so they never reach equilibrium. 8

9 Metabolic flux The rate of turnover of molecules through a metabolic pathway is known as metabolic flux. During different cellular conditions the rate of turnover can be different. Therefore the regulation of metabolic flux is important for all metabolic pathways to regulate each pathway under different conditions and ensure that the cellular processes are meeting the demands. The work of a biochemist spans many industries and functions. They may develop new medical treatments or devise new methods for brewing beer. Whatever their role, the biochemist s main purpose is to study the chemical processes of living organisms and to use their knowledge to develop or improve products and processes. Clinical biochemists look into the changes or faults of metabolic processes, slowing down the production of ATP for instance. In this case they would be concentrating on the enzymes needed during glycolysis, the link reaction, the Krebs cycle and the electron transport chain. Further reading Boyle, M. & Senior, K. (2008) Biology, 3rd Edition, HarperCollins Campbell, M.K. & Farrell, S.. (2011) Biochemistry, Cengage Learning Kennedy, P., Sochacki, F. & Hocking, S. (2008) CR Biology AS, Heinemann (Pearson Education Limited) Kennedy, P., Sochacki, F., Winterbottom, M. & Hocking, S. (2008) CR Biology A2, Heinemann (Pearson Education Limited) Moran, L., Horton, R., Scrimgeour, G., Perry, M. & Rawn, D. (2011) Principles of Biochemistry (International Edition), 5th Edition, Pearson Acknowledgements The publisher would like to thank the following for their kind permission to reproduce their photographs: Getty Images: Martin McCarthy / E+ All other images Pearson Education We are grateful to the following for permission to reproduce copyright material: Figure Electron transport chain from: http/ etc.png. Used by permission of Clarke Earley, Kent State University. In some instances we have been unable to trace the owners of copyright material, and we would appreciate any information that would enable us to do so. About the author Joanne Hartley studied Forensic Sciences at UCLAN and is currently the Science Vocational Coordinator, teaching KS3 and KS4 Science, Level 3 Forensic and Medical science and A-Level in a Merseyside school. She has been a Standards Verifier for four years, quality assuring vocational science courses across the country. Joanne has experience of authoring and examining, making contributions to the BTEC Level 3 Applied Science Student Book and Schemes of Work for the GCSE 2011 science. 9