Study Guide for Exam 3
Study Guide for Exam 3 BMB401
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Fructose 01/03/2016 ▯ Phosphorylation 1. Where does fructose comes from? Sucrose 2. Where does sucrose comes from? Fruit, sweet 3. Everything we eat gets absorbed in the gut and then what comes from the gut goes straight to the liver first. 4. The liver it absorbed a lot of glucose but with respect to galactose or fructose, all of it stays in the liver. 5. Fructokinase converts fructose to fructose 1-phosphate using ATP as the phosphate donor. 6. Fructose 1-phosphate is not phosphorylated to fructose 1,6-bisphos- phate as is fructose 6-phosphate, but is cleaved by aldolase B (also called fructose 1-phosphate aldolase) to dihydroxy-acetone phosphate (DHAP) and glyceraldehyde. 7. Humans express three aldolases, A, B and C, the products of three different genes. 8. Aldolase A (found in most tissues) 9. aldolase B (in liver, kidney, and small intestine) 10. aldolase C (in brain) all cleave fructose 1,6-bisphosphate produced during glycolysis to DHAP and glyceraldehyde 3-phosphate (see p. 100), 11. but only aldolase B cleaves fructose 1-phosphate into two components. 12. DHAP can directly enter glycolysis or gluconeogenesis, whereas glyceraldehyde can be metabolized by a number of pathways. Metabolism and Disorders 1. There are two things that could happen to glyceraldehyde. It can get phosphorylated by Triose kinase to get glyceraldehyde 3-phosphate into glycolysis. 2. Also Glyceraldehyde can get reduced to glycerol by alcohol dehydrogenase. 3. Glycerol can be phosphorylated to be glycerol 3-P by glycerol kinase. 4. Glycerol 3-P is used to make lipids (phosphoglycerides and triacylglycerols) 5. If you have a mutation in fructose kinase, its really not a big deal because the fructose wont get phosphorylated so it will stay in the blood and as soon as it gets to the kidney, fructose is taken out in the urine. (Fructosuria is the disease called) 6. The problem is when you have a problem with aldolase B isozyme. In this case fructose gets phosphorylated but then nothing can happen to it after, so its stuck inside the cell and it cannot be metabolized or do anything. It will end up building up in the cell and be toxic to the cell. This is in the liver so you will have all the outcomes that comes with liver disease. The disease is called Fructose Intolerance. Its survivable because you just don’t eat any fructose. Sperm Nutrient 1. Aldose reductase reduces glucose, producing sorbitol. 2. Sorbitol dehydrogenase oxidize the sorbitol to produce fructose. 3. The two pathways from glucose to fructose in the seminal vesicles benefits sperm cells, which use fructose as a major carbohydrate energy source. 4. In lenses, nerves and kidneys, the sorbitol has osmolites which is a compound that binds to water, they like to hydrate themselves, so if you have a bunch of sorbitol here, they are going to bring water into the cell and they become dysfunctional. 5. This is why diabetics loss kidney function, gets cataracts and have tingling in their nerves. Galactose metabolism and Disorders 1. Where does galactose comes from? Lactose 2. Where does lactose comes from? Dairy, milk 3. Galactose gets phosphorylated in the liver by galactokinase to galactose 1-P. 4. Galactose 1-P reacts with UDP-glucose and they kind of exchanged and you end up with UDP-galactose and Glucose 1-Phosphate by Galactose 1- Phosphate uridylyltransferase enzyme. 5. What can you do with UDP-Galactose? In this case is converted to glucose. 6. What is galactose? It’s the C-4 epimer of glucose 7. UDP-hexose 4-epimerase takes UDP-galactose and converts it into UDP- glucose (used to make glycogen) 8. Galactose is used in glycolipids, glycoproteins and glycosaminoglycans 9. Galactokinase deficiency is about the same thing not so harmful because it will go to the blood and then urine and its easy not to eat any dairy products. 10. Galactosemia is the one that is more recognized. It is galactose 1- Phosphate uridylyltransferase deficiency. It’s the one that its first screened in newborns because the first thing that will go to a baby’s mouth is milk. It causes diarrhea, vomiting and jaundice. Galactose 1-phosphate builts up on the cell and brings water and will have all the symptoms of liver disease. ▯ Pyruvate Kinase Inactivation 1. Pyruvate kinase converts PEP to pyruvate 2. PKA shuts off or inactivates pyruvate kinase by phosphorylation which decreases the conversion of PEP to pyruvate, which has the effect of diverting PEP to the synthesis of glucose. Pyruvate Carboxylase, PEP Carboxykinase 1. Gluconeogenesis is going on all the time. 2. We switch off pyruvate kinase and now we need to get the pyruvate that’s there to loop it up back to PEP. 3. You don’t have to be starving for gluconeogenesis to occur, its happening all the time because your red blood cells do not have mitochondria so your red blood cells are making ATP by glycolysis then the pyruvate gets converted to lactate acid and that regenerates the NAD+ that can go back up to the glyceraldehyde triphosphate dehydrogenase. What happens is that you are constantly making the lactic acid. 4. The other thing is the muscle, vigorously exercising muscle will make excess lactic acid. 5. The lactic acid is always coming to the liver filtered out of the blood from the muscle and red blood cells. 6. When lactic acid gets to the liver gets reconverted to the pyruvate. 7. You got to get this pyruvate out of the mitochondria to the cytosol so that it can become PEP. 8. Pyruvate carboxylase has a biotin prosthetic group and its biotin dependent. Carboxylates pyruvate to oxaloacetate. 9. The group carrier by Biotin is CO2 10. The CO2 goes onto the pyruvate generating oxaloacetate. 11. Oxaloacetate cannot cross the mitochondrial membrane so it is reduced to malate that can. 12. In the cytosol, malate is re-oxidized to oxaloacetate, which is oxidatively decarboxylated to phosphoenolpyruvate by PEP carboxykinase. 13. The enzyme in the cytosol is not the same enzyme because its an isozyme. PFK-2 Inactivation, FBP-2 Activation and FBP-1 Activation 1. High glucagon/insulin ratio causes elevated cAMP and increased levels of active protein kinase A. 2. Increased protein kinase A activity favors the phosphorylated form of the bifunctional PFK-2/FBP-2. 3. Phosphorylation of the PFK-2 domain inactivates it allowing the FBP-2 domain to be active. 4. Decreased levels of fructose 2,6-bisphosphate decreases the inhibition of FBP-1, which leads to an increased rate of gluconeogenesis. 5. Glucagon, your starving activates the PKA, which phosphorylates this PFK-2 which means that is shut off. 6. Glucagon stops glycolysis, stops glycogen biosynthesis. Fructose 1,6-Bisphosphate 1. Once you get to fructose 6-phosphate it goes to glucose 6- phosphate which then would go to the ER and the phosphatase will be here to take off the glucose 6-phosphate and make glucose to get secreted. Glucose 6-Phosphatase 2. Dephosphorylation of glucose 6-phosphate allows release of free glucose from the liver and kidney into blood. Gluconeogenic precursors include the intermediates of glycolysis and the tricarboxylic acid cycle, glycerol released during the hydrolysis of triacylglycerols in adipose tissue, lactate released by cells that lack mitochondria and by exercising skeletal muscle, and α-keto acids derived from the metabolism of glucogenic amino acids (Figure 10.10). Seven of the reactions of glycolysis are reversible and are used for gluconeogenesis in the liver and kidneys. Three reactions are physiologically irreversible and must be circumvented. These reactions are catalyzed by the glycolytic enzymes pyruvate kinase, phosphofructokinase, and hexokinase. Pyruvate is converted to oxaloac-etate and then to phosphoenolpyruvate (PEP) by pyruvate carboxylase and PEP-carboxykinase. The carboxylase requires biotin and ATP and is allosterically activated by acetyl coenzyme A. PEP- carboxykinase requires GTP. The transcription of its gene is increased by glucagon and the glucocorticoids and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6-phosphate by fructose 1,6- bisphosphatase. This enzyme is inhibited by elevated levels of AMP and activated when ATP levels are elevated. The enzyme is also inhibited by fructose 2,6-bisphosphate, the primary allosteric activator of glycolysis. Glucose 6-phosphate is converted to glucose by glucose 6-phosphatase. This enzyme of the endoplasmic reticular membrane is required for the final step in gluco-neogenesis as well as hepatic and renal glycogen degradation. Its deficiency results in severe, fasting hypoglycemia. Tissues 1. Where are the main stores of glycogen found? in skeletal muscle and liver, although most other cells store small amounts of glycogen for their own use. 2. What is the function of muscle glycogen? To serve as a fuel reserve for the synthesis of adenosine triphosphate (ATP) during muscle contraction. 3. Particularly during the early stages of a fast, what does the liver glycogen maintains? The blood glucose concentration. 4. Muscle makes glycogen and it uses it to supply its own energy. 5. The liver takes care of the brain 6. Liver makes glycogen to supply the blood with glucose in times when we have not eaten a meal and we are starving. UDP-Glucose 1. What is Glycogen? amylopectin 2. What is Amylopectin? α-1,4’ glucose chain and α-1,6’”branching” and its plant. 3. Phosphoglucomutase catalyzes convertion of Glucose 6- phosphate into a glucose 1-phosphate Glycogen Synthase 1. Glucose 6-phosphate becomes glucose 1-phosphate and this is reversible back and forth and no regulation. 2. UDP-glucose pyrophosphorylase catalyzes UDP-glucose from UTP and this catalysis releases PPi and pyrophosphatase with the addition of water forms 2Pi. 3. UDP-glucose is the sugar donor. 4. How do you start Glycogen biosynthesis? There is a protein called Glycogenin that is where this will originates. 5. Glycogenin is the autocatalytic enzyme adds glucose onto itself and that gets recognized by the glycogen synthase enzyme which adds glucose one after the other all the way down. 6. Glycogen synthase polymerizes alpha(14) glucose polymerization. 7. The Branching enzyme is called the 4:6 transferase and it makes the alpha(16) which is the branch and there are further elongation at the nonreducing ends by glycogen synthase, making alpha(1 4) bonds and further branching making alpha(1 6) bonds to make Glycogen. 8. We have branching so we can have many terminal ends that can be released. Glycogen phosphorylation 1. When your blood glucose goes down, your pancreas gets a signal and synthesizes and releases this hormone called Glucagon opposite of Insulin. 2. The Glucagon is a stress hormone, very similar to adrenaline and this binds to the GPCR and it will activate adenylyl cyclase which makes the cAMP which activates the protein kinase A. 3. Protein kinase A will result in phosphorylation of glycogen phosphorylase 4. Glycogen phosphorylase will release Glucose 1-phosphate 5. Protein kinase A does not phosphorylates glycogen phosphorylase directly. 6. Glycogen phosphorylase is phosphorylated by Glycogen phosphorylase Kinase. 7. During muscle contraction, Calcium is released from the sarcoplasmic reticulum. 8. Calcium binds to the calmodulin subunit of phosphorylase kinase b, activating it without phosphorylation. 9. Phosphorylase kinase can then activate glycogen phosphorylase, causing glycogen degradation. 10. Calcium does not need a stress hormone to activate phosphorylase kinase. Glycogen Synthase 1. Glycogen synthase makes glycogen 2. Glycogen phosphorylase degrades the glycogen. 3. Glucagon is a stress hormone that says that you are starving and that you need to release glucose and this activates protein kinase C results in phosphorylation. So if you phosphorylate the glycogen synthase, it turns it off because you want glucose. 4. The regulation of glycogen synthase activates the phosphorylase. 5. PKA inactivates the synthase and activates the phosphorylase. 6. cAMP comes from stress. 7. Insulin is going to do the opposite because when you eat you have lots of food and your pancreas secretes insulin that will bind to insulin receptor tyrosine kinase activates the IRS, activates the PI3K which phosphorylates PI(4,5)P2 to make the PI(3,4,5)P3 which recruits PDK1 and protein kinase B (PKB). 8. PKB is the protein kinase that phosphorylates and activates all these phosphatases. 9. Phosphatases undo all done by the Protein Kinase A (PKA) 10. for glycogen synthase, the active form is dephosphorylated, whereas the inactive form is phosphorylated by protein kinase A. 11. In the well-fed state, glycogen synthase b in both liver and muscle is allosterically activated by glucose 6-phosphate. 12. In contrast, glycogen phosphorylase a is allosterically inhibited by glucose 6-phosphate, as well as by ATP in both muscle and liver. 13. In the Liver, Glycogen phosphorylase is uniquely inhibited by glucose. 14. In muscle, glycogen phosphorylase is activated uniquely by AMP. Glucose Release Deficiency on Glycogen Storage Diseases 1. There is glycogen biosynthesis and there is glycogen degradation. 2. You could have a mutation on any enzyme and that’s called “enzyme deficiency” and it can be deficient for many different reasons. 3. What happens to a patient with the deficiency of the ability to make or degrade glycogen? These people have to eat every hour for the rest of their life to supplement their bodies with sufficient glucose to survive, even at nights. 4. If you have a problem with storage or the release of glycogen, you can always use gluconeogenesis to make glucose. Von Gierke Disease (glucose 6-phosphatase deficiency): this is because the enzyme is the last enzyme that takes glucose 6-phosphate to make glucose that will be released into the blood. If you lose this enzyme, you will not be able to released any glucose on your blood. Figure 8.11 ▯ Glucose is now in the cell and there is an energy investment phase where we are going to use 2ATP to put 2 phosphates on glucose. In the Energy generation phase, we will give those 2ATP back up and we are going to split the glucose into 3 carbon compounds and we ultimately we are going to get 2 pyruvates. ▯ Pyruvate: 3 carbons alpha kidoacid. ▯ At the end glucose a 6 carbon sugar becomes 2 pyruvates (2, 3 carbons compounds) ▯ The net will be of two molecules of ATP. We use two but we will going to get back 4, so the net production is 2 ATP. We also produce some NADH which can be used to produce some more ATP. ▯ ▯ Figure 8.12: Glucose 6-Phosphate ▯ Glucose become phosphorylated by an enzyme called Herokinase and another called Glucokinase. They both catalyzed the same reaction that is the transfer of the gamma phosphate, the last one of ATP onto the C6 hydroxyl. Once that phosphate is put on glucose, its trapped and this means that its “irreversible phosphorylation of glucose”. ▯ Hexokinase: is the enzyme in all of our tissue, brain, nerve, tissue, etc. ▯ Glucokinase: is an isoform of Hexokinase that is specific for the liver. ▯ ▯ Figure 8.13 Difference between Hexokinase and Glucokinase ▯ Glucokinase: so we just ate a big bowl of rice and there is a lot of glucose on that rice and what happens is that all that glucose comes through the liver which does a control check. The liver holds back a lot of the process of glucose after digestion and in doing so it has to process a lot of it. So Glucokinase has a HIGH Vmax, it can phosphorylate many glucoses per time. But it also has a HIGH Km value which means that it doesn’t bind glucose very tighly and having this high Km means that has a lower affinity for glucose. ▯ Hexokinase: in contrast they have a much lower Vmax but a very low Km and this is because there is not as much glucose to be process by these other tissues and having this low Km means that has a higher affinity for glucose, which means that is able to spot trace amounts of glucose in the cell. ▯ Figure 8.14 Glucokinase Regulation ▯ Glucokinase is indirectly inhibited by fructose 6-phosphate, which is in equilibrium with glucose 6-phosphate, a product of glucokinase and is indirectly stimulated by glucose (a substrate of glucokinase). ▯ Glucose has a chaperone and that is GKRP which is Glucokinase regulatory protein in the liver which grabs it and has it inactive until glucose comes. ▯ ▯ Figure 8.15 Fructose 6-Phosphate ▯ Reaction number 2: glucose 6-phosphate is isomerized to fructose 6-phosphate by phosphoglucose isomerase. ▯ Phosphoglucose isomerase is an aldo-keto-isomerase. There is an equilibrium between both substances. ▯ ▯ Figure 8.16 Fructose 1,6-Bisphosphate ▯ Fructose 6-phosphate is phosphorylated by an enzyme called phosphofructokinase-1 (PFK-1) so now we have fructose 1,6- Bisphosphate. This enzyme is inactive by itself and it need to be activated by Fructose 2,6-bis-phosphate. Bisphosphate means two phosphates but in different positions. After we have the Bisphosphate now comes Aldolase which splits the 6 Carbon compound into two 3 carbon compounds and they are Dihydrozyacetone phosphate and Glyceraldehyde 3-phosphate. Now we have the last enzyme here which is triose phosphate isomerase which catalyzes interconverting between DHAP and glyceraldehyde 3-phosphate. The DHAP must be isomerized to glyceraldehyde 3-phosphate for further metabolism by the glycolytic pathway. Figure 8.17 PKK-1 Regulation Fructose 6-phosphate must first be converted to fructose 1,6- bisphosphate. Insulin/glucagon are both secreted by pancreas and in response to feeding or starvation. Starvation: your glucose level goes down and brains send signal that we need more because it doesn’t want our muscles to burn all the fuel so it wants to stop this glycolytic process, to slow it down. So pancreas releases Glucagon which binds to GPCR activates adenylyl cyclase to make cAMP which activates protein kinase A which phosphorylates this PKF-2 and makes it inactive and without this you cannot make fructose 2,6-bisphosphate which is the one that activates PKF-1 to send fructose 1,6-bisphosphate down through glycolysis. Feeding: just ate and have lots of glucose so pancreas send Insulin binds to Insulin receptor tyrosine kinase which activates the PI3K kinase which then activates protein Kinase B and this activates the glucose transporters to go to the membrane and what it also does is that activates this enzymes called phosphatases and they hydrolyzes all of the phosphates created by the Glucagon signaling. The insulin activated protein phosphatase will remove the phosphate so dephosphorylated on the PFK-2 and when this happens the PFK-2 will become active and which favors formation of fructose 2,6- bisphosphate and will activate PFK-1 which leads to an increased rate of glycolysis. GLYCOLYSIS OCCURS IN THE CYTOPLASM Figure 8.18 NADH and ATP formation 1. Glyceraldehyde 3-phosphate conversion to 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction of glycolysis and NADH is formed. 2. When 1,3-BPG is converted to 3- phosphoglycerate, the high energy phosphate group of 1,3-BPG is used to synthesized ATP from ADP so it’s the first reaction where we actually form ATP. Catalyzed by phosphoglycerate kinase which is physiologically reversible. 3. Now we have 3-Phosphoglycerate where we move the phosphate from the 3 position to the 2 position by phosphoglycerate mutase which is freely reversible 4. From 2-phosphoglycerate we eliminate water by enolase to create a phosphoenolpyruvate. 5. Here is the second reaction where you form some ATP and again it is a substrate level phosphorylation. The conversion of PEP to pyruvate is catalyzed by pyruvate kinase (PK), the third irreversible reaction in glycolysis which makes ATP from ADP. The Fructose 1,6- bisphosphate ACTIVATES pyruvate kinase. 6. The side reaction that 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase. 2,3-BPG is present at high concentration in red blood cells and regulates Hemoglobin and serves to increase O2 delivery and its hydrolyzed by a phosphatase to 3-phosphoglycerate. Pyruvate is a 3Carbon alpha ketoacid. Figure 8.19 Pyruvate Kinase (PK) Regulation Its inactivated by Glucagon and it’s a switch point in glycolysis where you can turn glycolysis off in times of STARVATION. Active protein kinase A phosphorylates pyruvate kinase and inactivates it. Glucagon in times of Starvation will turn off PFK-2 and it turns off pyruvate kinase Figure 8.9 Aerobic and Anaerobic 1. The glycolytic pathway is employed by all tissues for the oxidation of glucose to provide energy (in the form of ATP) and intermediates for other metabolic pathways. (Figure 8.9A). Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars, whether arising from the diet or from catabolic reactions in the body, can ultimately be converted to glucose. 2. (Figure 8.9B) Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate supply of oxygen. This series of ten reactions is called aerobic glycolysis because oxygen is required to re-oxidize the NADH formed during the oxidation of glyceraldehyde 3-phos-phate Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the TCA cycle. 3. (Figure 8.9C) Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD+ .This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of oxygen. Anaerobic glycolysis allows the production of ATP in tissues that lack mitochondria (for example, red blood cells and parts of the eye) or in cells deprived of sufficient oxygen. Figure 8.21 Anaerobic Pyruvate is reduced to a hydroxyl to a (L)-Lactate. 1. In exercising skeletal muscle, NADH production exceeds the oxidative capacity of the respiratory chain. This results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intra-cellular pH and cannot make enough ATP on the muscle potentially resulting in cramps or burning. 2. Two molecules of ATP are generated each molecule of glucose converted to two molecules of lactate. THERE IS NO NET PRODUCTION OR CONSUMPTION OF NADH. Figure 8.20 PK Deficiency and Hemolytic Anemia The deficiency means that you are caring a mutation not that you DON’T have it. The symptoms to this is that people have Hemolytic Anemia (low red blood cell count, low hemoglobin and inability to carry out as much oxygen). This anemia observed in glycolytic enzyme deficiencies is a consequence of the reduced rate of glycolysis, leading to decreased ATP production. Mature RBCs lack mitochondria and are therefore completely dependent on glycolysis for ATP production. To fix Anemia they give you a blood transfusion. Figure 8.23 Insulin and Glucagon 1. The three irreversible reactions are Glucokinase, Phosphofructokinase and the Pyruvate kinase. The Brain can ONLY USE GLUCOSE TO FEED Figure 8.24 Control of Pyruvate 1. oxidatively decarboxylated by pyruvate dehydrogenase, producing acetyl coenzyme A. 2) carboxylated to oxaloacetate (a tricarboxylic acid cycle intermediate) by pyruvate carboxylase. 3) reduced by microorganisms to ethanol by pyruvate decarboxylase. ▯ Overview of Oxidative Phosphorylation 1. Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding what! CO2 and water 2. The metabolic intermediates of these oxidation reactions donate electrons to specific coenzymes which are… Nicotinamide Adenine Dinucleotide (NAD+) and Flavin Adenine Dinucleotide (FAD) 3. What do NAD+ and FAD form due to the oxidation reactions? the energy-rich reduced forms, NADH and FADH2. 4. These reduced coenzymes NADH and FADH2 can each donate a pair of electrons to a specialized set of electron carriers, collectively called … the electron transport chain (ETC). 5. As electrons are passed down the ETC, they lose what? much of their free energy. 6. The coupling of electron transport with ATP synthesis is called … oxidative phosphorylation 7. What’s OXPHOS? Oxidative phosphorylation. Mitochondrial Machinery 1. The Inner membrane of the mitochondria is impermeable to…most small ions, small and large molecules. 2. Matrix you have TCA cycle enzymes, Fatty acid oxidation enzymes, mtDNA, mtRNA and mitochondrial ribosomes. Electron Transport Chain 1. There are 4 Complexes in ETC 2. NADH electrons first comes into complex I and gets transfer to complex III and then Complex IV. 3. FADH2 electrons go to Complex II and then gets transfer to Complex III and then Complex IV. 4. Complex I: We have NADH that comes from the mitochondria and its the substrate that will be oxidized by dehydrogenases. 5. The name of Complex I is NADH Dehydrogenase 6. Dehydrogenases remove the electrons from NADH and transfer to FMN (flavin mononucleotide) also inside in the Complex I. 7. Succinate dehydrogenase: At Complex II, electrons from the succinate dehydrogenase–catalyzed oxidation of succinate to fumarate move from the coenzyme, FADH2, to an iron-sulfur protein, and then to coenzyme Q. 8. Coenzyme Q (CoQ) is a quinone derivative with a long, hydrophobic isoprenoid tail. 9. CoQ is a mobile electron carrier and can accept hydrogen atoms from NADH dehydrogenase (Complex I), from succinate dehydrogenase (Complex II), and from other mitochondrial dehydrogenases: glycerophosphate dehydrogenase and acyl CoA dehydrogenase. 10. CoQ transfers electrons to Complex III (cytochrome bc1) and then, links the flavoprotein dehydrogenases to the cytochromes. 11. Cytochromes: The remaining members of the ETC are cytochrome proteins. Each contains a heme group . 12. Cytochrome C is not a complex it just carries electrons from Complex II to Complex IV. 13. Electrons are passed along the chain from cytochrome bc1 (Complex III), to cytochrome c, and then to cytochromes a + a3 (Complex IV) 14. Complex IV is called cytochrome c oxidase because its being oxidized. 15. At Complex IV, the transported electrons, O2, and free protons are brought together, and O2 is reduced to water. 16. Complex II is Succinate dehydrogenase. 17. The TCA cycle is Complex II, its embedded in the membrane. 18. Complex III and Complex IV can only do one electron at the time 19. Why would electrons go through all these Complexes to form at the end water? That’s electro chemistry Energetic Coupling 1. Blocking electron transfer by any one of these inhibitors stops electron flow from substrate to oxygen because the reactions of the electron transport chain are tightly coupled like meshed gears. 2. There are compounds or antibiotics that specifically blocks oxidative phosphorylation and without them we would have never been able to figured this stuff out. 3. Electron Transfer Chain reaction occurs from compounds with high negative to compounds more positive. 4. The more negative the Eo of a redox pair, the greater the tendency of the reductant member of that pair to lose electrons. 5. The more positive the Eo, the greater the tendency of the oxidant member of that pair to accept electrons. 6. Electrons flow from the pair with the more negative Eo to that with the more positive Eo. 7. ½ O2/H2O is the most oxidant and the stronger oxidizing agent that can accept electrons. 8. NAD+/NADH is the strongest reducing agents that is that they have stronger tendency to lose electrons. ATP Synthase 1. Electron transport is coupled to the phosphorylation of ADP by the pumping of protons across the inner mitochondrial membrane, from the matrix to the intermembrane space, at Complexes I, III, and IV. 2. When NADH flows through Complexes I,III and IV, it gives you three NADH protons. 3. When FADH2 flows through Complex II, which is not a pump, but since electrons will go after this through Complex III and IV we will get two protons. 4. This process creates an electrical gradient and a pH gradient. 5. The energy generated by this proton gradient is sufficient to drive ATP synthesis. 6. The proton gradient serves as the common intermediate that couples oxidation to phosphorylation. 7. The multi-subunit enzyme ATP synthase (Complex V) catalyzes formation of ATP from ADP+Pi. 8. The ATP synthase contains a domain (Fo), and an extra- membranous domain (F1) 9. The F0 domain spans the inner mitochondrial membrane. 10. The F1 domain appears as a sphere that protrudes into the mitochondrial matrix. 11. Fo rotation causes conformational changes in the β subunits of the F1 domain that allow them to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. 12. The ATP/ADP transporter is able to move ADP to the matrix so it can react with the ATP synthase and all the ATP that will be formed moved to the outside. Uncoupling Proteins 1. Uncoupling proteins form channels that allow protons to reenter the mitochondrial matrix without energy being captured as ATP. 2. The energy is released as heat, and the process is called nonshivering thermogenesis. Glycerol 3-P and Malate 1. 2. The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol cannot directly enter the mitochondrial matrix. 3. However, two electrons of NADH are transported from the cytosol into the matrix using substrate shuttles. 4. In the glycerophosphate shuttle two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerophosphate dehydrogenase. 5. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme glycerol 3-phosphate as FAD is reduced to FADH2. 6. CoQ of the ETC oxidizes the FADH2. 7. The glycerophosphate shuttle, therefore, results in the synthesis of two ATPs for each cytosolic NADH oxidized. 8. There is a malate dehydrogenase on the outside and on the inside that can convert NADH on the outside to NADH on the inside. 9. These two shuttles yields three ATPs 10. What passes electrons through the mitochondria? Malate and Glycerol 3-phosphate Myopathy Neurodegenerative disease are dealing with patients who have weak mitochondria in their nerves or muscle and they are not able to make the ATP needed, then cells are unable to be robust and run propertly. PDH Complex (Pyruvate dehydrogenase complex): 1. It’s a complex that contains three enzymatic activities, which are Pyruvate decarboxylase, Dihydrolipoyl transacetylase and Dihydrolipoyl dehydrogenase. 2. It has five different coenzymes, thiamine pyrophosphate (TPP), lipoic acid, CoA, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD+) 3. What comes in and what comes out? Pyruvate comes in to the intermediate in the mitochondria from the cytoplasm and acetyl CoA comes out to the matrix of the mitochondria. 4. Pyruvate, 3 carbons alpha-keto acid reacts with pyruvate decarboxylase and thiamine and in doing so is decarboxylated, it results in the formation of CO2, the acetyl group get transfer to coenzyme A which that’s going to feed into the TCA cycle, and the electrons that came from hydroxyethyl are transfer to FAD, FADH2 and FADH2 release them to NAD+ to NADH. 5. NAD+ comes from the mitochondria. Regulation of PDH complex 1. The PDH Complex is the gateway from the cytosol to the mitochondria. 2. When you make too much ATP you got to turn it off and the way to do this is that there is an enzyme called PDH kinase will catalyzed phosphorylation of the PDH Complex and it will turn off. 3. Substances don’t regulate the PDH Complex, they do regulate the PDH kinase and then the Kinase will regulate the Complex. 4. What activates the PDH Kinase? Generally the products of the pathway when you have plenty of this product. 5. What are those products that activate the PDH Kinase? Lots of NADH and Acetyl CoA and ATP 6. What does inactivate PDH Kinase? Lots of Pyruvate. 7. What do Phosphotases hydrolyzes off? the phosphate. 8. What activates the PDH Complex again? PDH Phosphotase 9. What activates the PDH Phosphotase? By Calcium released during contraction in skeletal muscle. Formation of α-ketoglutarate from acetyl coenzyme A (CoA) and oxaloacetate 1. We need to know the reaction and this means that there is a substrate and a product and then what kind for reaction is it and what it’s the enzyme that catalyze the reaction. 2. Citrate synthase is the enzyme that catalyzes the condensation of acetyl CoA and oxaloacetate and water hydrolyzes off the CoA. 3. What is created from the condensation of Acetyl CoA and oxaloacetate? A tricarboxylic acid that have 6 carbons, which is Citrate. 4. Citrate is isomerized to isocitrate by which enzyme? Aconitase 5. What is Aconitase? An Fe-S protein. 6. What does Isocitrate dehydrogenase catalyzes? the irreversible oxidative decarbozylation of Isocitrate 7. What does Isocitrate dehydrogenase yields when catalyzes Isocitrate? 3 NADH and the first release of CO2. 8. What does the catalysis of Isocitrate generates? α-ketoglutarate. 9. Alpha-ketoglutarate is a 5 carbon alpha-keto dicarboxylic acid. 10. The enzyme Isocitrate dehydrogenase is activated by what? ADP ( a low-energy signal) and Calcium. 11. What regulates or inhibits the enzyme Isocitrate dehydrogenase? Elevated levels of ATP and NADH. Alpha-Ketoglutarate Dehydrogenase (NADH, CO2) into Succinate Thiokinase (GTP) 1. What catalyzes the conversion of alpha-ketoglutarate to succinyl CoA? Alpha-ketoglutarate dehydrogenase complex. 2. The mechanism to convert alpha-ketoglutarate to succinyl CoA is an oxidative decarboxylation. 3. What does Alpha-ketoglutarate dehydrogenase complex yields? A second CO2 and a second NADH for the cycle. 4. What will regulate α-ketoglutarate dehydrogenase complex? Its products which are too much NADH and succinyl CoA 5. What will activate α-ketoglutarate dehydrogenase complex? Calcium. 6. What cleaves the high-energy thioester bond of succinyl CoA? Succinate thiokinase . 7. The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation. 8. Succinate is oxidized to fumarate by which enzyme? succinate dehydrogenase 9. What’s a 4 carbon alpha-keto dicarboxylic acid? Succinate 10. FAD (its coenzyme) is reduced to what when succinate dehydrogenase oxidazes succinate and makes a double bond? FADH2. 11. Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded where? in the inner mitochondrial membrane. 12. Fumarate is hydrated so adds the water to L-malate in a freely reversible reaction catalyzed by which enzyme? fumarase 13. L-Malate is oxidized to oxaloacetate by which enzyme? malate dehydrogenase. 14. Which reaction produces the third and final NADH of the cycle? L- Malate being oxidized by malate dehydrogenase to oxaloacetate. Regulation of the Cycle 1. the most important of the regulated enzymes in the TCA cycle are those that catalyze reactions with highly negative ∆G0 and they are? citrate synthase, iso-citrate dehydrogenase, and α- ketoglutarate dehydrogenase complex. 2. Reducing equivalents needed for oxidative phosphorylation are generated by what? the PDH complex and the TCA cycle 3. both processes of PDH Complex and TCA cycle are up-regulated in response to a decrease in the ratio of ATP to ADP.
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