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Lecture

General intermediary metabolism.docx

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Department
Biochemistry
Course
BCH3120
Professor
Elaine Beaulieu
Semester
Winter

Description
General intermediary metabolism Lecture 2 If we were to be at equilibrium, we would be a gas ball, cause we would be at equilibrium with air which wouldn’t work bc all of our biomolecules would dissipate. Our body works to keep its complexity so we are in a dynamic steady state. your body will not start making bits that they don’t need unless its energy that will be stored. It will only make what is needed. Proteins have half-lives, in different tissues, different proteins have different functions. So in liver that half-life is 0.9 days so the proteins are being laid down quickly. So virtually, your whole body is being re made. The need for energy is constant. In the universe there is always the same amount of energy, it can only be transformed. Nutrients in the environment are what heterotrophs use in order to create complex molecules. So we transform complex molecules into simple molecules. We also have a little bit of heat, we need this to maintain our body temperature. There is less entropy inside our body (more order) and more entropy outside our body (less order). We oxidize glucose and that’s how we make our energy. The electron flow is derived from nucleophiles and electrophiles. Metabolic pathways – carbohydrate metabolism is the main source of energy, all products stem from this. In segregation, lysozymes will be exported for example to keep them away from the reaction and degrading it. Selective transport is used on the cell membrane. Mitochondria are the only place where the Krebs cycle occurs and also oxidation phosphorylation. Certain organelles contain the proper enzymes to undergo metabolic pathways. Insulin will be secreted and will have a diff or similar function and will induce certain pathways in different organs. A lot of the time, enzymatic reactions will not have all the things necessary for thermodynamic to go ahead but some of these have so much in them so they will drag along the ones that are not thermodynamic – domino effect. Energy is transformed in many ways – work. Entropy tends to be spoken of as complex molecules being transformed to simple molecules. Free energy is what is available from either a reaction or pathway. The reaction won’t go forward when it is endergonic (delta G > 0). The delta G of the hydrolysis of ATP is negative so it is thermodynamically favorable. Standard free energy is a constant. The reactions act together to drag one another forward, giving a sum of negative for the total free energy. The intermediate that links energy releasing catabolic reactions with energy demanding anabolic reactions is ATP. All that is released will be used by ATP and stored for the use of breaking down complex molecules into simple molecules. Enzymes reduce the energy that it needs for the reaction to occur so that they can occur with lower energy. Inhibition occurs when an inhibitor binds to the substrate region on the enzyme and stops the reaction. Multiple things can bind to an enzyme and alter its capacity. Or it can alter in such a way that will open the substrate binding site pocket so that there is more accessibility for the substrate. Glycerol can bind to 4 places on its enzyme (glycerol kinase). Large amount of ATP inhibits catabolic pathways. Glucose and fructose can bind glycerol kinase and will either tell it to convert into G3P or will stop the reaction. All the covalent modifications are reversible. When a protein has a PEST motif, it is automatically sent for ubiquitinization and is destroyed, depending on the PEST sequence they can survive for 2 to 3 mins or a while longer Lecture 3 - Glycolysis We use carbohydrates as fuel. Aldose – a carbonyl group that is found at the end of the carbon chain. Ketose – a carbonyl group that is found attached to a carbon that is not found at the end of the chain. As humans, our bodies use alpha stereoisomers, whereas plants use beta which incorporates cellulose. Glucose can be used in several ways. It can be used as storage and be stored as glycogen, starch and sucrose. It can undergo oxidation via glycolysis which creates pyruvate which will be used in the krebs cycle. It can also undergo oxidation via pentose phosphate pathway and become ribose-5-phosphate which is important in order to generate nucleic acids. And finally it can be used in the extracellular matrix and in cell wall polysaccharides when it there is synthesis of structural polymers. Transporters are needed to pump glucose from outside the cells to inside the cell – bi directional. Glut 5 is special because it is a fructose transporter. GLUT4 exported through the membrane via insulinodependant transport – insulin needs to bind to its receptor, the receptor will phosphorylate p85 which is a subunit of a PI3 kinase complex. This will activate p110 which will convert PIP2 into PIP3. PIP3 will recruit AKT which needs to be phosphorylated by 2 kinases. Once it is phosphorylated, it stops AS160 from converting Rab-GDP into Rab-GTP which means that Rab-GDP cannot segregate GLUT4 into the cytoplasm anymore and will be transported to the membrane. In the feeder pathway, hexokinase is a very important enzyme; it can transform several molecules so that they can be used in the glycolysis pathway. There are 2 major processes in anaerobic glycolysis. We have the investment phase and the payoff phase. In the investment phase, step 1 is a major metabolic control point, meaning that 1 ATP is used and the reaction cannot be reversed. Here, we have the phosphorylation into glucose-6-phosphate. The advantage of this step is that it has no choice but to stay into the cell, we`ve trapped it; this also helps maintain a gradient, so that more glucose from outside of the cell would want to come into the cell. The enzymes responsible for this step are hexokinase and glucokinase which each have their own activity. Glucokinase is glucose specific and is only found in the liver and the pancreas; it has a very low affinity to its substrate (glucose). Hexokinase is not specific to glucose, so it can use fructose or mannose. It has a strong affinity for its substrate and is allosterically regulated by its product (glucose-6-phosphate). These 2 enzymes have different enzymatic activity. Hexokinase which is found in your muscles runs very quickly on very little glucose. Glucokinase is the opposite; it requires a lot of glucose to reach max activity. Step 3 is another metabolic control point, it is irreversible and is thermodynamically favorable and is a limiting step. Step 9 – PEP is a high energy molecule. Step 10 is the last metabolic control point, it produces an ATP and is irreversible. Because glucokinase is not inhibited by G6P means that it can keep creating more G6P. For hexokinase, in our muscles, the substrate which is G6P will inhibit the reaction; therefore our muscles will not make more glycolysis than it needs to. So it will make enough and when it has enough, it will stop making it. Whereas in the liver, the glucose that is made will be stored so we don’t want hexokinase to stop working. We want it to keep working so that G6P can keep working so we can store the extra glucose as glycogen. Glucokinase also differs from hexokinase in such a way that when there is low glucose, what you really want is the glycogen to be broken down into glucose. The hexokinase will be segregated into the nucleus so you can stop that from happening so that the glucose can come out. The F6P will drive glucokinase to be segregated into the nucleus by its regulatory protein. Step 3 is regulated by phosphofructokinase-1. Its controlled allosterically and it will be driven forward when there is low energy (AMP and ADP) and also by fructose bisphosphate. But it will be inhibited by citrate which is a metabolite from the krebs cycle. Therefore when the krebs cycle is going, citrate it being produced and its telling glycolysis that it doesn’t need to make more pyruvate. In the same way, when ATP is high, you want to stop glycolysis bc you have enough energy. Finally, it is activated by low energy molecules. Step 10 is the last step of glycolysis; it is where PEP is converted into pyruvate kinase. This is controlled allosterically as well; it is inhibited by acetyl- CoA and ATP because these are products of the krebs cycle, therefore if there is sufficient amount, then there will have a negative feedback control. Insulin and glucagon have opposite effects in the cell. Under aerobic conditions, the pyruvate from glycolysis will undergo fermentation. Pyruvate is changed into 2 lactates when your body undergoes vigorous activity. During lactic fermentation, the coenzyme NAD+ is anaerobically reoxidized by fermentation in the cytosol, whereas lactic fermentation occurs in the muscles and the red blood cells. So pyruvate, through lactate dehydrogenase, will be transformed into lactate which will be able to use the NAD+ to regenerate NAD which is necessary for the glycolysis to go through. During lactic fermentation, the store of glucose in your muscles will all have been eaten up and you need oxygen in order to undergo gluconeogenesis and to restore the glycogen stores. Cori cycle – the glycogen is used by the muscles which produces ATP, the lactate will take up the blood circulation and will be transformed into glucose in the liver which requires an ATP, the glucose will be able to take back the blood cycle where it will either be used or stored as glycogen. Lecture 4 – Gluconeogenesis and the pentose phosphate pathway Gluconeogenesis is the synthesis of new molecules of glucose or glycogen from non- carbohydrate sources. In order to do the inverted pathway, we have to circumvent the irreversible steps by using different enzymes. Pyruvate will be transformed back into phosphoenolpyruvate and then reverse the glycolysis pathway all the way to fructose-16- biphosphate where fructose-16-biphosphotase will transform this back into fructose-6- phosphate and then in order to circumvent the step 1, glucose-6-phosphotase will transform G6P back into glucose. If you’ve been fasting for greater than a day and a half, the gluconeogenesis in the liver will be the only source of glucose in your body, you’ll have depleted all your glycogen. If an average person needs 150g of glucose per day, and your liver can nly produce 100 then something is missing… Only from lactate, glucogenic amino acds, and glycerol can be transformed back into glucose. Also, glucogenic amino acids can by transformed directly into pyruvate. Pyruvate carboxylase is only present in the mitochondria. So pyruvate has to be shuffled into the mitochondria in order to be transformed into oxaloacetate. OA cannot get out of the mitochondria, therefore it has to be shuffled through a mechanism where it will be transformed into malate which can go into the cytosol which is transformed back into OA. And then malate can be transformed in OA in the cytosol via malate dehydrogenase. This is called the malate shuttle. Now that we have OA, it can be transformed into phosphoenolpyruvate via PEP carboxylase. The rest of the steps follow the reversal of glycolysis, so we have the change of phosphoenolpyruvate  2-phosphoglycerate 3- phosphoglycerate  1,3-bisphosphoglycerate  glyceraldehyde-3-phosphate and then dihydroxyacetone will be used to condense the whole into fructose-1,6-biphosphate. This will be converted into fructose-6-phosphate via fructose-1,6-bisphosphatase. This will now be transformed into glucose-6-phosphate which will finally be transformed into glucose via hydrolysis by glucose-6-phosphotase. This is a very costly reaction, it uses 4 ATP, 2 GTP and 2 NADH, but it’s physiologically necessary. The suns in the picture represent where the energy rich molecules are being taken up. Gluconeogenesis from lactate is seen in the Cori Cycle. Gluconeogenesis from alanine: alanine is produced during the catabolism of amino acids in the muscles. The transfer of amino group onto alpha-ketoglutarate is the first step. This will then be transferred into glutamate. Glutamate will be transformed into alanine which will then be transformed into pyruvate in the liver. Pyruvate will then undergo gluconeogenesis. This is called the alanine cycle and it is only used under rare conditions such as hyperproteic diets, fasting, and type 1 diabetes. So alanine is then transported to the liver from the muscles via the blood, once in the liver it is transformed into pyruvate via 2 transamination processes. The pyruvate undergoes gluconeogenesis to make glucose and then goes where it’s needed. Gluconeogenesis from glycerol: glycerol comes from triglyceride hydrolysis in the kidneys and the liver. Glycerol is changed into glycerol 3 phosphate via glycerol kinase. Then glycerol-3- phosphate will be changed into dihydroxyacetone phosphate and this can be condensed into fructose-1,6-bisphosphate which will be converted into glucose. Gluconeogenesis from glucogenic amino acids: amino acids can be transformed into krebs cycle precursors, such as tryptophan into alanine and threonine, and these into pyruvate which will be transformed into acetyl-coA and used in the krebs cycle. The point is that there are different entry points. All the amino acids are glucogenic except for leucine and lysine. The pentose phosphate pathway is not designed to produce energy. It allows the production of NADPH which is used in the anabolism of fatty acids, so it’s needed in various reducing steps. It also produces ribose-5-phosphate which is the skeleton for nucleic acids. The liver uses very little glucose, it gathers its energy from the fatty acids. The substrate for PPP is glucose-6- phosphate and produces F6P and G3P. The first step is a 2 enzymatic step. First, the oxidation phase, it’s the oxidation of G6P into 6-phosphogluconate, this step is irreversible and used G6P dehydrogenase and lactonase. Step 2, the isomerization phase, is the oxidation and decarboxylation of the 6-phosphogluconate into ribulose-5-phosphate. This is then epimerized into xylulose-5-phosphate by ribulose-5-phosphate epimerase. Now, during the non-oxidative phase, these 5 carbon molecules will be changed in turn to form F6P and G3P. So, a 5C molecule will be transformed briefly into a 7C molecule to generate a 6C molecule (fructose-6- phosphate) and a 4C molecule. Now, xylulose-5-phosphate will be transformed in order to generate another fructose-6-phosphate and a G3P. During this pathway, you end up using 6 xylulose-5-phosphate and ribulose-5-phosphate in order to generate 5 6C molecules that will be transformed into glucose-6-phosphate. Looking at the non-oxidative phase with more detail, step 4, xylulose5P and ribulose5P undergo a transketolisation to become glyceraldehyde3P and sedoheptulose7P. Step 5 uses a transaldolase taking these intermediates and changing them into fructose6P and erythrose4P. Step 6 is another transketolisation which changes erythrose4P and xylulose5P into fructose6P and glyceraldehy
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