CBI 7: Glycolysis and Gluconeogenesis 2024/25 PDF

BMB Year 1 CBI 7: Glycolysis and Gluconeogenesis 2024/25 In this session, we will study some key metabolic pathways relating to intracellular energy transfer. The production of energy in the form of ATP is mainly performed by the cell in a series of connected processes: glycolysis, oxidat...

BMB Year 1 CBI 7: Glycolysis and Gluconeogenesis 2024/25 In this session, we will study some key metabolic pathways relating to intracellular energy transfer. The production of energy in the form of ATP is mainly performed by the cell in a series of connected processes: glycolysis, oxidative decarboxylation, the TCA cycle, and oxidative phosphorylation. All these processes have lots of steps so let's start with the first one - glycolysis - and its reverse reaction gluconeogenesis... WELCOME Welcome to CBI 7 COR E CON TEN T Glycolysis Gluconeogenesis Energy balance and regulation KEY TER MIN OLOGY AN D AB B R EVIATION S  Key Terminology R ECAP AN D R EADIN ESS Knowledge check Summary and further reading Section 1 of 7 Welcome to CBI 7 BMB Year 1 Welcome to CBI 7 Maintenance of glucose homeostasis is critical for survival. Dysregulation of plasma glucose concentrations is a characteristic of diabetes, a disease that is reaching epidemic proportions around the world. Introduction to energy balance and glycolysis There are too many metabolic pathways for us to look at all of them in detail. Therefore we are going to focus on those that are central to energy production and energy balance. Let's start with the aerobic respiration of glucose. The overall process we are looking at starts with glucose as a carbon source, generating water and carbon dioxide. In doing so, the energy released is captured through processes that then allow the ATP to be produced. The aerobic cellular respiration of glucose is summarised below. Overall, one molecule of glucose and six molecules of oxygen are used to form six molecules of carbon dioxide and six molecules of water: C6H12O6 + 6 O2→ 6 CO2 + 6 H2O If this reaction happened all in one step, we would call this combustion, and clearly, no ATP would be produced. In cellular respiration, a series of enzyme-catalyzed reactions allow the chemical potential energy of substrates to be harnessed to power ATP production. There are four main sets of metabolic processes to consider which we will look at in turn in the subsequent sections: Glycolysis Oxidative decarboxylation TCA/Krebs cycle Oxidative phosphorylation and electron transport chain VIMEO CBI 7 Introduction This video gives an overview of what will be covered in the pre-session emodule of CBI 7. VIEW ON VIMEO  Transcript Introduction CBI 7.pdf 179.9 KB C O NT I NU E Learning objectives Please read through the following list of intended learning outcomes for both this eModule and the face-to-face session. After studying this eModule, you should be better able to: 1 Provide a concise definition for each of the terms in the Key Terminology highlight box 2 Explain the different phases of glycolysis, their purpose and the locations in the cell where it takes place. 3 Describe why gluconeogenesis is not simply the reverse reaction of glycolysis and how gluconeogenesis circumvent irreversible reactions of the glycolysis. 4 Describe the interplay of glycolysis and gluconeogenesis in maintaining energy balance. In this eModule, you will be asked to review material that will give you the background knowledge to read and understand any recommended reading material and prepare you for the face-to-face session. Image source: www.gemmacorrell.com Section 2 of 7 Glycolysis BMB Year 1 Steps in glycolysis First, we are going to look at glycolysis. As the name suggests, this is a set of reactions that result in the lysis (breaking) of glucose. In fact, the reactions in glycolysis result in two molecules of pyruvate being produced per single molecule of glucose that is metabolised. Glycolysis also results in the reduction of two molecules of NAD+ to form two molecules of NADH, and an overall net production of two molecules of ATP from ADP. So, overall, the reaction looks like this: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O Glycolysis takes place in the cytosol and can be divided into three major steps comprising a total of ten enzyme-catalyzed reactions. Let’s take a look at an overview (below) first before we dive deeper into detail.  The 10 chemical reactions that take place during glycolysis. The whole process can be divided into three major steps: the investment phase, break down into two C 3-fragments and the payoff phase. Adapted from Stryer 2018 by Dr. Silke Kerruth. There will be a lot of chemical structures in this eModule. We do not expect you to learn any of these structures or enzyme names by heart but as this is a chemistry module and the chemical reactions that are taking place are fairly simple we wanted to include this detail here. Ok, let's have a look at the chemical reactions in each step and their purpose: Step 1: Trapping glucose and preparing it to be cleaved The first step of the glycolysis contains three enzyme-catalysed reactions and is also called the investment phase as energy is consumed in the form of ATP to generate fructose-1,6- bisphosphate. VIMEO  Glycolysis - Step 1 This video introduces the reactions of the first step during glycolysis, also called the investment phase. VIEW ON VIMEO  Transcript Glycolysis Step 1.pdf 88.9 KB Why does adding a phosphate group prevent glucose from leaving the cell? It becomes too bulky.  The negative charge prevents it from diffusing through the membrane via the glucose transporter. The addition of the phosphate leads to the fast conversion into fructose-6-phosphate before it can diffuse out of the cell. The concentration of glucose-6-phosphate is much higher outside the cell so it does not diffuse against the concentration gradient. SUBMIT C O NT I NU E Step 2: Lysis into two C3-units The second step of glycolysis splits the fructose-1,6-bisphosphate into two different C3 units. If each of these underwent a different pathway to be converted to the end product (pyruvate), more enzymes would be necessary which would mean a waste of resources for the cell. Thus one enzyme converts dihydroxyacetone phosphate into glyceraldehyde-3- phosphate.  VIMEO Glycolysis - Step 2 In the second step of glycolysis fructose-1,6-bisphosphate gets cleaved into two C3 units that get further metabolised in step 3. VIEW ON VIMEO  Transcript Glycolysis Step 2.pdf 79.3 KB The isomerisation of dihydroxyacetone phosphate into glyceraldehyde-3- phosphate is a reversible reaction. How is the equilibrium of this reaction shifted towards glyceraldehyde-3-phosphate?  The change in Gibbs free energy favours the formation of glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate is used up by the next reactions in the glycolysis pathway. The equilibrium is not shifted towards glyceraldehyde- 3-phosphate but towards dihydroxyacetone phosphate. The equilibrium is shifted by energy input via the hydrolysis of ATP to ADP. SUBMIT C O NT I NU E Step 3: The payoff phase Up to this point, there has been no net energy produced from the reactions in glycolysis, in fact two ATP molecules were consumed in step 1 (the investment phase). The third step of glycolysis is also called the payoff phase as it produces two times two molecules of ATP and two times one molecule of NADH. NADH is also used to produce ATP via oxidative phosphorylation which will be covered in CBI 8. VIMEO Glycolysis - Step 3 In the third step of glycolysis glyceraldehyde-3-phopshate is converted into pyruvate. This step is also called the payoff phase as energy is released via ATP and NADH production. VIEW ON VIMEO  Transcript Glycolysis 3.pdf 84.2 KB Which of the following reactions describes a substrate level phosphorylation? Transfer of a phosphate group from ATP to another molecule. Transfer of a phosphate group from one carbon atom to another in the same molecule. Transfer of a phosphate group from a molecule to ADP in order to form ATP. Formation of ATP from ADP and Pi using a proton gradient across the membrane. SUBMIT So, in summary, we can see that through a combination of activating phosphorylation steps, glucose is lysed, and then each of the lysed components used to generate two molecules of pyruvate, along with a net production of 2 ATP and 2 NADH. 9Remember, in the investment phase two molecules of ATP were used up, while during the payoff phase, two molecules of ATP were produced. However, the payoff phase happens twice per molecule of glucose, giving a net production of two molecules of ATP. C O NT I NU E The fate of pyruvate Pyruvate is the final product of glycolysis. However, depending on the state of the cell, and the organism, it can be converted into different products. Under normal aerobic conditions the three-carbon compound pyruvate will be converted into the two-carbon compound acetyl-CoA (not considering the coenzyme A group). During this reaction one molecule of CO2 is released. Acetyl-CoA can then be further oxidised in the TCA or Krebs cycle. This part will be covered in CBI 8. However, the steps of glycolysis are not dependent on oxygen. The metabolism of glucose in the absence of oxygen is called fermentation. Fermentation takes place in all organisms but can form different products. For example, yeast (and several other microorganisms) can convert pyruvate to ethanol, while in the human body pyruvate gets converted into lactate.  Different pathways by which pyruvate is used up. Ethanol and lactate are the end products of fermentation, which happens under anaerobic conditions. Normally, most of the pyruvate gets converted into acetyl-CoA and enters the Krebs/TCA cycle. C O NT I NU E Other simple sugars In our diet we consume lots of different sugars and not only glucose. For example in CBI 5 you learned that sucrose is a disaccharide made from glucose and fructose, while lactose (milk sugar) is made of glucose and galactose (see image from CBI 5 below to freshen your memory). Those disaccharides are split in our body into their building blocks so that each monosaccharide is then converted to either glucose-6-phosphate or other components within the glycolysis pathway. Alternatively, these sugars can be converted to glycogen for storage. Important disaccharides in our diet: sucrose and lactose. Section 3 of 7 Gluconeogenesis BMB Year 1 Gluconeogenesis: synthesis of glucose Glucose is the most important substrate for energy metabolism in our body. The brain accounts for the largest amount of glucose needed during the day (about 120 g, "Stryer: Biochemistry, 9th edition, p. 519"). Thus, our body needs to maintain a constant level of glucose. This is secured by the synthesis of glucose from non-carbohydrates, a process called gluconeogenesis. Importantly, this term/process should not be confused with glyconeogenesis, which is the production of glycogen. You learned about glycogen in CBI 5 which is a macromolecule made of glucose molecules in order to store glucose in cells. Make a note of the spelling, but more importantly, recognize these two processes are very different indeed! Earlier in the eModule, we examined the steps in glycolysis, shown again below. Gluconeogenesis is almost the reverse sequence of steps to glycolysis, but not quite. Note: We will use the abbreviation GNG for gluconeogenesis in this session. Where does gluconeogenesis occur? All good so far, but where is this happening? Surely all cells in the body would like to be able to do this, gluconeogenesis trick, right? Nope! As it happens, GNG only occurs in two organs in the body:  Liver Adrenal cortex (kidneys) One obvious consequence of this organ specificity is that glucose produced by GNG will have to travel through the blood stream to supply the glucose needed in other organs (i.e. the brain).  All the reactions in the glycolytic pathway are bidirectional except three reactions that are effectively only able to proceed in one direction; these are highlighted in the figure above. Why these three? The reason why these reactions are effectively irreversible lies in the change in Gibbs free energy (ΔG, see CBI 4: Biomolecular Reactions). For these three reactions, the value of ΔG is very negative, so they are highly exergonic. In order to revert those reactions, a lot of energy would be needed, but this is not available in these systems for these individual enzymatic steps, and therefore we observe that these enzymes only catalyze these reactions in one direction under normal conditions. Gibbs free energy changes in glycolysis The enzyme-catalyzed reactions in glycolysis are each associated with a change in Gibbs free energy, as shown on the right. This clearly presents an issue for gluconeogenesis, as we have three reactions that simply won't proceed in the reverse direction that is required for them to convert pyruvate to glucose. What have organisms developed through evolution to overcome this? How is it done? As you have probably guessed, an alternative set of enzymes is used to bypass/reverse these steps, using other biochemical processes. Let's take a look at these enzymes as they are critical to GNG in relation to the effectively irreversible steps in glycolysis they bypass. Glycolysis - Step 1 The forward reaction in glycolysis catalysed by hexokinase while the reverse reaction used in gluconeogenesis is catalysed by glucose-6-phosphatase, a membrane bound protein in the membrane of the endoplasmatic reticulum (ER). Thus, this reaction does not take place in the cytoplasm but in the lumen of the ER. Glycolysis - Step 3 The forward reaction in glycolysis is catalyzed by phosphofructokinase. The reverse reaction used in gluconeogenesis is catalyzed by fructose-1,6-bisphosphatase. Glycolysis - Step 10 The forward reaction in glycolysis is catalyzed by pyruvate kinase. The reverse reactions used in gluconeogenesis is catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEP-CK). This actually requires two separate enzyme-catalyzed reactions. First pyruvate is converted into oxaloacetate, an important molecule of the TCA/Krebs cycle we will revisit again in the next CBI session. This reaction takes place inside the mitochondrial matrix. Then oxaloacetate is converted into malate, which is the only way to transport oxaloacetate from the mitochondrial matrix into the cytoplasm (via the malate-aspartate-shuttle). In the cytoplasm, malate is converted back into oxaloacetate, and then decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase. Note: These two reactions require energy in the form of ATP and GTP. To form glucose we need two molecules of pyruvate so these energy-consuming reactions must take place twice. C O NT I NU E GNG Summary The overall equation for GNG, starting with pyruvate as a substrate and catalyzed by the reactions outlined above, is as follows: 2 Pyruvate + 4ATP + 2GTP + 2NADH + 6H2O --> Glucose + 2NAD+ + 4ADP + 2GDP + 6Pi + 2H+ The two GTP and two ATP molecules are consumed when reversing the 10th step of glycolysis (2 pyruvate -> 2 phosphoenolpyruvate). The other two molecules of ATP are consumed in the reverse step of the 7th reaction of glycolysis. This step included a substrate level phosphorylation where a phosphate group was transferred to a ADP molecule. Here in the reverse reaction energy is used by the hydrolysis of ATP to form the energy-rich compound of 1,3-bisphosphoglycerate. Again this step needs to take place twice in order to form one molecule of glucose. Alternative GNG precursors We have considered GNG process with pyruvate as the starting substrate, but there are a number of additional substrates that can feed into the process and allow the de novo synthesis of glucose: Lactate (skeletal muscle) Glucogenic amino acids (all except leucine and lysine) Glycerol (lipolysis) C O NT I NU E Section 4 of 7 Energy balance and regulation BMB Year 1 Energy balance Our bodies are always working to maintain our energy homeostasis - i.e. to ensure that the processes in the cell/body can balance the flow of (bio)chemical energy between inputs (e.g. from nutrients/foods) and energy expenditure (i.e. work done). To maintain this balance, the activity of different biochemical pathways/processes need to be regulated depending on what is needed and when. In simple terms, we are talking about the balance between catabolic reactions (that break down molecules into smaller subunits and release energy), and anabolic reactions (that require energy to synthesise complex molecules from simpler/smaller subunits). Anabolism is the opposite of catabolism. Energy balance between catabolism and anabolism For example, glucose synthesis is an anabolic process, whereas the breaking down of glucose is a catabolic process. Glucose is a good example as it is one of the main dietary sources of energy; as we have seen already, it can be used as a substrate for glycolysis and subsequent reactions to generate ATP in both the presence and/or absence of oxygen. Whereas glycolysis (as its name indicates) is the breakdown of glucose, the process of gluconeogenesis refers to its synthesis de novo (meaning: 'from new / from the beginning'). It is also worth mentioning here that glucose monosaccharide units can be linked and stored (in the form of the macromolecule polysaccharide glycogen; structure discussed in CBI 5) in the liver, which is effectively a longer-term energy store that can be utilized when required. C O NT I NU E Blood glucose We introduced the idea that the body regulates biochemical pathways to ensure energy homeostasis. It is understandable that we want to ensure that we don't run out of ATP and therefore ensuring dietary intake and de novo production of glucose is necessary, but why worry about having too much? Basically, our bodies need to maintain blood glucose levels within a normal range, typically between 4-8 mM, termed normoglycaemia. As can be seen from the graph, after a typical meal, (also referred as the postprandial period, or postprandial state), blood plasma glucose levels increase as it is absorbed (hyperglycaemia), and then decreases as glucose is subsequently taken up by different cells/organs. The glucose taken up by cells can be used as a substrate for glycolysis. Glycolysis is therefore a catabolic pathway that will be switched on when we have high levels of glucose in our blood and/or we need to use it for other processes. Starvation glucose levels There will be times that we don’t have that much glucose on our bloodstream (hypoglycaemia), (i.e during starvation or when energy demand has driven glucose uptake and utilization), so there is a need to produce glucose to be used as a source of energy. During starvation, our bodies still need to maintain blood glucose concentration within the physiologically normal range to maintain healthy function. OK! So, what happens then? Well, if glycolysis is switched on when glucose levels are high it is reasonable to predict that when glucose levels are low, it will be switched off and some other process that produce glucose (i.e. gluconeogenesis, the production of glucose) - will be switched on. Glucose will be produced and released to the blood to maintain the concentration within the normal range. C O NT I NU E Regulation of glucose levels You have seen how critical it is to maintain a stable blood glucose level. Thus, our body has different mechanisms to regulate the processes that are involved in the usage (glycolysis) and production (gluconeogenesis) of glucose. There are two main regulation processes that are differ in their time frames and approaches. Fast regulation It would make little sense for a cell to have both glycolysis and gluconeogenesis running at the same time. Thus, some of the enzymes that are involved in these processes are critical points when it comes to the regulation of these processes in the cell. It is easy to imagine that the concentration of the products and substrates will have an effect on which of these metabolic processes will take place. Secondly, the concentration of ADP or ATP are effectively a measure of the energy availability in the cell, and therefore also important. The fastest way for the cell to regulate glycolysis and gluconeogenesis is by inhibiting or activating enzymes involved in these processes. Only two steps are strongly regulated: Slow regulation In the liver the rates of glycolysis and gluconeogenesis control the blood-glucose level, thus each pathway is strictly controlled by hormones. If blood glucose levels fall then the peptide hormone glucagon is released from the alpha cells of the pancreas. Glucagon activates gluconeogenesis but also the conversion of stored glycogen into glucose. In contrast, when blood-sugar levels are high, then the beta cells of the pancreas release the peptide hormone insulin that promotes glycolysis. These hormones work mainly by altering the expression of the genes that encode for the enzymes used in glycolysis or gluconeogenesis. Thus, this type of regulation is much slower than the allosteric inhibition we have described above. C O NT I NU E Section 5 of 7 Key Terminology BMB Year 1 Key terminology You should be able to provide a concise definition for the following terms: Glycolysis The metabolism of glucose to produce pyruvate with one molecule of glucose being consumed, and two molecules of pyruvate being produced, alongside the reduction of two molecules of NAD+ to form two molecules of NADH, and overall net production of two molecules of ATP from ADP Gluconeogenesis (GNG) The de novo synthesis of sugars (typically glucose) from metabolic precursors Investment phase (of glycolysis) Steps in glycolysis that require the input of energy, provided by the hydrolysis of ATP Payoff phase (of glycolysis) Steps in glycolysis that produce energy in the form of ATP and NADH Substrate level phosphorylation A phosphate group is transferred from a high energy substrate to ADP to form ATP Normoglycaemia The condition in which there is a normal concentration of glucose in the blood. Hypoglycaemia The condition in which there is a concentration of glucose in the blood lower than the range considered normal Hyperglycaemia The condition in which there is a concentration of glucose in the blood higher than the range considered normal Section 6 of 7 Knowledge check BMB Year 1 Here are a few questions to see what you can remember about the eModule content. Question 01/08 In the first reaction of glycolysis glucose is phosphorylated to glucose-6-phosphate. What is the purpose of this phosphorylation? Activating glucose for splitting into fragments. Trapping glucose inside the cell. Production of ATP via substrate level phosphorylation. Adding a negative charge for transport into the mitochondria. Question 02/08 With respect to the reactions in glycolysis, which of the following statements is correct? Glucose-6-phosphate is split to form two molecules of glyceraldehyde- 3-phosphate Glucose-6-phosphate is split to form one molecule of glyceraldehyde-3- phosphate and one molecule of dihydroxyacetone phosphate Fructose-6-phosphate is split to form one molecule of glyceraldehyde- 3-phosphate and one molecule of dihydroxyacetone phosphate Fructose-1,6-bisphosphate is split to form one molecule of glyceraldehyde-3-phosphate and one molecule of dihydroxyacetone phosphate Question 03/08 What kind of reaction takes place on the glyceraldehyde-3-phosphate substrate in the picture shown below? Phosphorylation reaction Oxidation reaction Reduction reaction Dehydration reaction Question 04/08 Look at the reaction shown below. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses this reaction. How does the enzyme avoids the formation of a low energy intermediate? Phosphorylation of the intermediate Metal ions inside the enzyme stabilse the intermediate Formation of a thioester intermediate Reaction takes place in one step instead of two. Question 05/08 Under anaerobic conditions pyruvate gets converted into which molecule in the human body? Acetyl-CoA Ethanol Lactate Oxaloacetate Question 06/08 There are ten enzyme-catalyzed reactions in glycolysis. The change in Gibbs free energy for these reactions is shown in the table. Seven steps are reversible. Use the data available to identify which three steps are effectively irreversible. Reactions 1, 2, and 3 Reactions 1, 3, and 10 Reactions 7, 8, and 9 Reactions 2, 4, and 6 Question 07/08 The last step of gluconeogenesis is the conversion of glucose-6-phosphate to glucose. In which compartment of the cell does this reaction take place? Cytoplasm Mitochondrial matrix Nucleous Lumen of the endoplasmic reticulum Question 08/08 What is the name given to lower than normal blood glucose levels? Hypoglycaemia Normoglycaemia Anemia Hyperglycaemia Section 7 of 7 Summary and further reading BMB Year 1 Learning Objective Checklist After studying this eModule, you should be able to: Provide a concise definition for each of the terms in the Key Terminology highlight box Explain the different phases of glycolysis, their purpose and the locations in the cell where it takes place. Describe why gluconeogenesis is not simply the reverse reaction of glycolysis and how gluconeogenesis circumvents irreversible reactions of the glycolytic pathway. Describe the interplay of glycolysis and gluconeogenesis in maintaining energy balance. There is a lot of detail here, and a common question is whether everything needs to be committed to memory. In a word - no.  While it is sometimes useful to have all these details memorized (e.g. exact order of all enzymes and their substrates/products) this is not a good use of time and effort at this stage. Some people like memorizing such things, in which case great - they are at liberty to do so! Most people will simply rely on notes and literature to check these things when they need to. What is more important is to appreciate what each of the processes does, and the relevance of the inputs and outputs from each step. It is more important that you appreciate you know how things work, rather than remembering long lists of names and numbers. At this point, just think about the big players (glucose, pyruvate, acetyl CoA, NADH, FADH2) and why they are relevant. The upcoming hybrid face-to-face session will give us the opportunity to look back over this eModule. Overall, we will aim to explore the interplay of glycolysis and gluconeogenesis in maintaining energy balance, and the enzymes responsible for catalyzing related biochemical reactions. We will take the opportunity to start integrating what we have learned across CBI so far to better understand how energy balance is regulated by different biochemical mechanisms. Further Reading If you want to read more about glycolysis, all the steps are explained with further details about the chemical reactions taking place in "Chemistry for the Biosciences" (pages 447-456). Additionally, the chapter in Stryer's "Biochemistry" focus a bit more on the enzymes involved in each step (pages 491-505); gluconeogenesis is also described in detail (pages 519-524). eModule Feedback On the BMB we are committed to providing you with the best learning experience possible. Please help us to achieve this by answering our short questionnaire about this eModule. Thanks! CLICK HERE Any questions? Please post any questions/comments you have about the content to the CBI Discussion Board.