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CHEM345-Biochemistry Final Exam Fall 2007 HONOR CODE: 1. Using the some (does not have to be all) of the following vocabulary words, explain the importance of integration in metabolism. Discuss common reaction themes. DO NOT DEFINE EACH SEPARAETLY, rather define within your answer. This should be a summary of metabolism. THIS SHOULD ONLY be 1-2 page of material. (30pts) ATP NADH Acetyl CoA Dehydrogenases Phosphate pyruvate glycogen glucose CO2 and O2 glutamate oxidation/reduction phosphoryl transfer fatty acids compartmentalization The overall goal of metabolism is to supply the body with energy, metabolites, and macromolecules to carry out proper function and maintain homeostasis. Although carbohydrate metabolism is the central pathway in all metabolism, the anabolic and catabolic pathways of other macromolecules (lipids/fatty acids, proteins, and nucleic acids) follow the same guidelines and have similar pathways to supply the body with what it needs. Metabolism is coordinated based on the energy needs (ATP/ADP ratio) of the cells as well as the blood glucose level (glucose, a carbohydrate, is the primary energy source of the body). It is imperative that blood sugar be maintained to keep a constant supply of glucose to the brain, the largest consumer of glucose in the body. Through enzymes called dehydrogenases, carbohydrates are oxidized to release high energy electrons from their carbon-carbon bonds, reducing electron carriers (NAD+ → NADH, FAD→FADH2), which carry electrons to the electron transport system to be used in the production of ATP by oxidative phosphorylation when the cells are working in the low energy state (low ATP/ADP ratio). Glucose is provided from the diet (circulating through the blood) or glycogen stores in the liver, along with gluconeogenesis in the liver. When glucose levels are low and glycogen stores have been depleted from the liver, and gluconeogenesis in the liver is no longer effective in producing sufficient amounts of glucose, fatty acids and amino acids are degraded to resupply glucose to maintain blood sugar (important for brain function) and also to generate other intermediates that can be funneled into the TCA cycle to liberate high energy electrons, but can come at higher costs to the body, primarily the brain and kidneys. Although degradation of fatty acids can harbor lots of energy potential, under starving conditions, degradation could be deadly. Under starving conditions, the brain is deprived of glucose, and when glucose is scarce, fatty acids are converted via acetyl CoA to ketone bodies, which temporarily replace glucose. When fatty acids are degraded in excess, these ketone bodies create an acidic environment in the body, leading to acidosis and disrupting homeostasis, and leading to organ malfunction. This is also true of excess amino acid degradation. When amino acids are degraded, they release glutamate, which contains a carbon-carbon backbone to generate glucose. To salvage this backbone, the amine group is released, creating urea. If too many amino acids are degraded, this leads to increased amounts of urea, which is toxic to the body, especially the kidneys. Under high ATP levels, the body does not need to produce excess ATP, so oxidative pathways to release electrons are inhibited and glucose and other metabolites are stored. Glucose can be reconverted from various metabolites that are in excess via gluconeogenesis and excess glucose is stored in the liver as glycogen. If there are still excess amounts of metabolites and metabolic intermediates, then they are stored as fatty acids and lipids. For example, in the storage of fats, excess levels of acetyl CoA are converted to cholesterol, a lipid molecule used to maintain membrane fluidity. Essentially, pyruvate, glucose6-phosphate, and acetyl CoA are the central molecules of metabolism because they are versatile and can be funneled through many processes based on what is needed in the cell. Pyruvate is probably the most flexible because it can be converted to enter numerous catabolic as well as anabolic pathways, and can come from several different sources, as well as act as an allosteric inhibitor. It is the product of glycolysis, which can be used to fuel the TCA cycle under low ATP conditions by being converted to acetyl CoA; it can be reconverted to glucose in times of high energy charge, and when ATP does not need to be made (primarily in the liver); it is a primary molecule in the lactate salvage pathway. It is also a convergence point for other macromolecular metabolic process, such as lipids/fatty acids and amino acid metabolism. In fatty acid degradation, glycerol can be sent to the liver and converted to pyruvate via glycolysis, which can it can be used to produce glucose by gluconeogenesis. During the degradation of proteins, numerous amino acids can be degraded to pyruvate, either directly or indirectly. And the reversal is true also – pyruvate can be degraded in order to help anabolize lipids and macromolecules as well. Some of these aspects of pyruvate can be said for acetyl CoA as well. Acetyl CoA can be produced readily from every macromolecule via degradation, but cannot be reconverted quite as easily. Acetyl CoA cannot be used to regenerate glucose via gluconeogenesis because it cannot be reconverted to pyruvate. So when acetyl CoA enters the mitochondria, it is subject to numerous fates of either further catalysis or to fatty acid/lipid synthesis, depending on what the cell needs and signals from the brain. Glucose-6-phosphate is an important component of the pentose phosphate shunt, a series of side pathways that generates glycolytic intermediates for glycolysis and the production of pyruvate, as well as intermediates for other pathways, as well as ribose-5-phosphate (for the production of nucleotides). The production and funneling of metabolic intermediates is one of the most important aspects of metabolism. During degradation, intermediates from fatty acid and amino acid degradation can be funneled into TCA so they can be utilized for ATP production, or these intermediates can be funneled out of TCA in the production or storage of macromolecules. The fates of intermediates are dependent on location (compartmentalization), energy charge of the cell, and blood sugar level. If a particular intermediate is in one location with the cell being at a certain state, it is subject to one type of reaction, while in another state, it may need to be funneled somewhere else to perform another function. 2. Using figures in your as guides text, predict what would happen in 3 organ systems under starvation (low sugar) conditions. How does Diabete mimic this situation. (HINT: remember signaling and what’s on, what’s off and why?) 30pts Under starvation conditions, blood sugar (blood glucose) levels are low and the energy charge of the cell is low. The brain is the body’s largest consumer of glucose, and when deprived, it begins to malfunction. Under these circumstances, the brain (particularly the adrenal medulla) secretes glucagon in response to low glucose, which travels via the circulatory system to the liver (which can be referred to as the “powerhouse” of metabolism because it performs numerous metabolic activities) to degrade glycogen. Glucagon binds and facilitates a GPCR mediated response, which stimulates a cAMP signal transduction cascade, activating protein kinase A and, through a series of phosphorylations, ultimately activating glycogen phosphorylase a. This enzyme catalyzes the cleavage of a single glucose molecule off of glycogen (via phosphorylation) and glucose enters the blood, being delivered primarily to the brain to supply it with the energy it needs, and some travels to the tissues as well to generate ATP necessary for function. Gluconeogenesis is also on in the liver to generate glucose from free intermediates. The liver can only store a day’s worth of glycogen, so when these stores get depleted, the brain is once again deprived of the glucose it needs, so the body turns to fat degradation. The adipose tissue houses triacylglycerols, which harbor great energy potential. In ultimate starvation, these triacylglycerols are hydrolyzed to their glycerol and fatty acid components in the adipocytes. Then, the glycerol can be funneled directly to the liver and converted to glucose via gluconeogenesis, which then gets supplied to the brain by the blood. Fatty acids can be directed to other cells, where they are converted to acetyl CoA and enter the TCA cycle to release electrons and generate ATP. However, the glucose from glycerol may not be sufficient enough to supply the brain with all the energy it needs, and as glucose levels keep getting smaller, the brain is more and more deprived, and the fatty acids keep getting degraded. Fatty acids produce acetyl CoA when oxidized, but acetyl CoA cannot be used to regenerate glucose (cannot enter gluconeogenesis). Instead, in the liver (within the mitochondria of liver cells), acetyl CoA gets converted to ketone bodies, which can be utilized by the brain in place of glucose for a short while, but are insufficient. If acetyl CoA keeps being degraded into ketone bodies due to high levels of fat degradation, there will be an increased level of ketone bodies circulating through the blood and subsequently to the brain. Increased levels of these ketone bodies causes blood pH to drop, which disrupts homeostasis and leads to metabolic and organ dysfunction. The brain is the organ that is affected the most by the pH drop. Malfunction of the brain in ultimate starvation results in death. Diabetes is a reflection of this process because in diabetes, there is very little to no insulin production, which is needed for the uptake of glucose from the blood. Instead, this leads to increased levels of glucagon circulating through the system. Increased glucagon leads to overstimulation of PKA and glycogen phosphorylase, which causes increased amounts of glucose in circulation and increased glycogen depletion, with decreased amounts of uptake by the cells, and less utilization of glucose for energy. The increased glucagon circulation leads the body to believe that it is in starvation because glucose is not being taken up by the cells. Once these stores are depleted, triacylglycerols are hydrolyzed at a faster rate, releasing increased amounts of fatty acids and subsequently greater production of ketone bodies. Excess ketone bodies leads to decreased pH in the blood, resulting in acidosis and subsequently diabetic coma and death. 3. Amino acids contribute to biochemistry is multiple ways. Choose two global functions that amino acids serve and describe in detail. (HINT: structure!!!) (20pts) Amino acids are the building blocks of proteins. They constitute the primary sequence of polypeptides, which are transcribed from genes in a DNA sequence into an mRNA strand. Sequences of amino acids in a polypeptide determine the folding of secondary structures, or structural motifs, known as alpha-helices and beta-pleated sheets. Secondary structures are determined by the hydrogen bonds between individual amino acids of the peptide that come together during folding. The R groups of individual amino acids are directed towards the outside of the structure, giving it special characteristics, such as charge, that allow it to have special interactions with the environment. For example, alpha-helices that have amino acids with hydrophobic side groups tend to be found in hydrophobic environments, such as the plasma membrane. These helices tend to be part of transmembrane proteins, where the transmembrane domain is made of hydrophobic helices. These secondary structures are only determined by pieces of the polypeptide based on sequence and structure of amino acids; in the end all of the resultant secondary structures come together to form the final tertiary (3D) structure based on the sequence of all the amino acids. This final structure is stabilized by noncovalent association between neighboring amino acids in the 3D structure. Certain amino acids have come together to form and stabilize the tertiary structure based on their electrochemical and physical properties. The tertiary structure of the protein determines its function. Therefore, the primary sequence of the polypeptide dictates the function of the protein. If there is an error in the primary sequence, then the protein does not fold correctly and results in a faulty, inactive protein. A major class of proteins is enzymes, which act as catalysts to speed up the rate of a spontaneous chemical reaction. Enzymes contain active 3D clefts that bind a particular substrate (specific for the enzyme), and catalyze a specific reaction (converting the substrate to a desired product). The amino acids that are located within the catalytic site are the agents that help drive a particular reaction forward; the shape and electrical environment of the catalytic site are dictated by the amino acids. The arrangements of the amino acids (their location in the primary sequences allow them to come together in the tertiary structure) as well as their electrical and chemical properties (electrostatic interactions, hydrogen bonding capabilities, van der Waals and hydrophobic interactions) contribute to the enzyme’s specificity for a particular substrate in a chemical reaction. Interactions between the amino acids of the catalytic site (known as the catalytic group) and the amino acids of the substrate allow for a perfect fit of the substrate into the site. Therefore, substrate shape and shape of the active site should be complementary. The electrochemical properties of the catalytic group facilitate the formation of the substrate’s transition state and subsequently the final product. For example, serine proteases (such as chymotrypsin) contain a “catalytic triad,” made up of a serine, a histidine, and an aspartate. The chemical characteristics of the triad amino acids orient each other to create a strong nucleophile in the catalytic site, which allows for sufficient cleavage of peptides via hydrolysis. The triad dictates the shape of the catalytic site so the enzyme can only cleave peptide bonds on the carbonyl side of amino acids that contain benzene rings, such as tryptophan and phenylalanine. 4. Eating a low carbohydrate/ high protein diet appears OK on the surface, but what happens if you take it to the extreme. Using what you know about pathway regulation and on/off switches explain what happens and why it can have a negative effect on the body overtime. (20pts) Eating a low carbohydrate/high protein diet can seem okay because it results in quick weight loss. But this loss is only short term, and prolonging this diet can have negative, even long term effects on the body. Essentially, the body thinks it is in starving conditions because there is low glucose (the primary energy source) circulating throughout, and less glucose is getting to the brain. So the brain sends a signal, glucagon, to the liver to degrade glycogen in order to produce glucose. Glygogen phosphorylase is activated from the protein kinase A signal transduction cascade, which cleaves single glucose molecules from glycogen. Glycogen synthase has been inactive by protein kinase A so as to not store glucose in the brain’s time of need. Gluconeogenesis involving other metabolites is occurring in the liver as well. Glucose that enters the muscles is entering glycolysis and cellular respiration to supply the muscles with energy in order to perform optimally. Most of the glucose, however, travels to the brain. Once glucose and glycogen stores have been depleted, the brain is still in constant need of energy, so the body turns to fat degradation as well as amino acid degradation; amino acids are in abundance because of increased levels of protein in the diet. Because fatty acids and amino acids are not the primary energy molecules for the human body, increased catabolism of these molecules can cause problem. The ingested protein gets digested to proteases in the small intestine, and amino acids are released into the blood stream. These amino acids are utilized for their carbon-carbon backbone to enter gluconeogenesis in the liver. In the liver, amino acids get converted to glutamate. In order to be utilized for energy, glutamate must be stripped of its amine group. This is done by glutamate dehydrogenase, in which glutamate gets oxidized to αketoglutarate and releasing an ammonium ion and NAD+ is reduced to NADH, which carries electrons to be carried to the electron transport system. Under low energy charge, the αketoglutarate can be funneled into TCA in the muscles to liberate high energy electrons. Or αketoglutarate can be funneled via the malate-aspartate shuttle to the cytoplasm, where it can be converted to oxaloacetate, a primary molecule in gluconeogenesis. However, this can only occur in the liver because cells are actively metabolizing glucose through glycolysis. The ammonium ion that is released is converted to urea via the urea cycle to leave the body because it is toxic. If there are increased levels of protein degradation, urea levels are increasing. Because urea is toxic, increased levels could have a serious impact on the body, primarily the kidneys. Because the body cannot replenish degraded proteins, and proteins cannot be stored, the body will only utilize enough amino acids so protein levels do not deplete. Thus the primary degradation pathway in times of starvation is utilization of fats. Triacylglycols, the fat energy storage in the adipose cells, are broken down into glycerol and fatty acids. Glycerol is transported via the blood to the liver, where it is reconverted to glucose by gluconeogenesis to be supplied to the brain, while fatty acids go through β-oxidation to produce acetyl CoA and can be funneled into the TCA cycle to release electrons and produce ATP in the muscles. However, when fat degradation is the primary metabolic pathway, there is decreased uptake of glucose by the muscles from the blood because of high glucagon/low insulin levels, and fatty acids can diffuse freely into the cell. There are increased levels of acetyl CoA in the cells from fatty acid degradation; the increased acetyl CoA acts as an allosteric inhibitor of pyruvate dehydrogenase, which stops the conversion of pyruvate to acetyl CoA. Thus, fatty acids become the major energy source for muscle cells. Because of this increase in acetyl CoA, glycolysis is turned off, and pyruvate can be sent to the liver to be converted to glucose by gluconeogenesis. The TCA cycle cannot keep up with the increased influx of acetyl CoA, so excess amounts of acetyl CoA are converted to ketone bodies in the liver and become the brain’s major source of fuel in the absence of glucose. The brain can survive a shortThis is an inefficient way to supply the brain with energy because increased levels of ketones circulating in the blood decreases the overall pH, leading to acidosis and sickness, and possibly death. 6. Higher order organisms have evolved multi-subunited enzyme systems to control metabolic pathways. Using one of the following a) describe the structure of the enzyme complex, b) explain how it functions, c) explain how it is regulated, and d) explain why such enzyme complexes arose. (20pts) You may use a diagram here. Fatty Acid Synthatase Pyruvate Dehydrogenase Pyruvate dehydrogenase catalyzes the irreversible reaction of pyruvate → acetyl CoA. It is irreversible because acetyl CoA cannot be converted back to pyruvate and subsequently glucose. a. Pyruvate dehydrogenase is a large complex whose quaternary structure is composed of three distinct enzymes - Pyruvate dehydrogenase component (E1) – α2β2 tetramer - Dihydrolipoyl transacetylase (E2) – catalytic trimer - Dihydrolipoyl dehydrogenase (E3) – αβ dimer The core of the complex is composed of 8 E2 enzymes (8 catalytic trimers) and each trimer has a flexible lipoamide arm. 24 E1 and 12 E3 enzymes surround the active E2 core. This close proximity increases the rate and efficiency of the reaction taking place and reduces the possibility of unnecessary side reactions. This close proximity allows for movement of the flexible lipoamide arm, which is a main component of the reaction process. b. Pyruvate dehydrogenase acts by performing a decarboxylation, a series of oxidation/reduction reactions involving cofactors, and transfer reactions in order to decarboxylate pyruvate and convert it to acetyl CoA. Each subunit performs a particular task in the overall reaction. E1, a dehydrogenase, performs an oxidative decarboxylation of pyruvate, releasing CO2, and creating an active intermediate containing the desired acetyl group with the use of the cofactor TPP. The lipoamide arm from E2 comes into the E1 site and the acetyl group is transferred to the lipoamide arm, and then returns to the active core. E2, a transacetylase, transfers the acetyl group to CoA, generating the desired product. But the lipoamide arm needs to be reoxidized in order to carry out the process again. So it swings into E3, a dehydrogenase, which gets oxidized by the FAD cofactor (which in turn gets reduced, carrying electrons) and is ready to repeat the cycle. The new FADH2 is then able to reduce an incoming NAD+ to generate NADH, which can be used to carry electrons to the ETS, and FAD is reoxided so it too can take part in the reaction once again. c. Pyruvate dehydrogenase is regulated by multiple allosteric regulations and covalent modification. Because it catalyzes an irreversible reaction, it is strongly subject to regulation based on the needs of the cell, and can be inhibited or activated when necessary. In times of high energy charge, (high ATP/ADP ratio) pyruvate dehydrogenase can be allosterically regulated by the products that it helps to create – acetyl CoA, ATP, and NADH. When these products are in abundance and do not need to be produced any more, they will bind to an allosteric site on the enzyme and inhibit or slow down the rate of the reaction it catalyzes because the cell does not need any more of the molecule. Under low energy conditions, the enzyme needs to be activated to produce acetyl CoA and subsequently ATP. It can be allosterically activated by ADP and pyruvate and even CoA because they are in abundance and need to be converted to help produce energy. Pyruvate dehydrogenase is also regulated by covalent modificiation. In times when the products are in excess, then enzyme does not need to continue catalyzing the reaction; phosphorylation of the E1 subunit by pyruvate dehydrogenase kinase inactivates the enzyme and slows down the rate of the reaction. PDK can be activated by the products of pyruvate dehydrogenase, which will then phosphorylate the enzyme to slow it down. When there is a need for these products, then the dehydrogenase will be dephosphorylated by its phosphatase, allowing it to produce product. d. 7. Regulation is a key aspect of Biochemistry. We have seen this over and over again. Choose one of the following topics and describe how “regulation” plays an important role. NOTE: We are not just talking about enzymatic control, but others levels of control too. (20pts) a. Protein Synthesis b. Protein Folding c. Compartmentalization Compartmentalization, or location within the cell, is a major control mechanism because it designates the fate of certain molecules and intermediates to specific metabolic pathways depending on their location in the cell (the mitochondria or the cytoplasm) as well as the energy needs of the cell. Molecules in one region of cell are subject to specific pathways than when they are in another region. Therefore, the funneling of intermediates between the mitochondria and the cytoplasm is tightly controlled. Under certain conditions, enzymes are allosterically regulated and covalently modified in order to service the needs of the cell. In times of high energy charge and the cell has certain metabolites in abundance, enzymes will be inactivated by the products it produces (allosteric inhibition) or by phosphorylation (covalent modification). Answer 1 (one) of the following: 8. You are a scientist at the New You Corporation. You are working hard to discover the protein involved in “anti-aging”. You know only the amino acid sequence of this protein and the fact that it binds to and catalyzes the addition of telomeres on the ends of genes (a process that halts aging in cells). a. In short answer suggest how you could purify this protein from a cell lysate. (10pts) b. Upon purification you find you don’t have enough of the protein to use for your assays. In short answer how could you make more of this protein? (10pts) c. What assay could you perform to determine that this protein is fully functional and ready to experimentation? (10pts) 9. Nothing is ever wasted in Biochemistry. Describe in detail two examples of this. Be sure to explain why this is important. (30pts) During periods of exercise and the cells are actively metabolizing, glucose gets converted to pyruvate via glycolysis. During periods of intense exercise, pyruvate is being produced at excess rates and oxygen is depleted quickly, creating an anaerobic environment. Instead of being reduced to acetyl CoA by pyruvate dehydrogenase, pyruvate is reduced to lactate by lactate dehydrogenase because pyruvate cannot enter the TCA cycle under anaerobic conditions. Lactate cannot be further oxidized in the cells to yield energy; however, it can be recycled in the liver to reproduce glucose. This process of salvaging lactate to regenerate glucose is called the Cori Cycle, and shifts the weight of metabolism from the muscles to the liver. In order to be metabolized further, lactate is channeled from the muscles to the liver by the circulatory system, where lactate gets reduced back to pyruvate, which then gets converted back to glucose by gluconeogenesis. This glucose is then funneled back to fuel the actively metabolizing tissues and resupply glycolysis. Lactate salvage allows the body to maintain a balance of glucose in the blood as well as the tissues. Glycolysis is responsible for making 4 ATP molecules; during this intense exercise, it helps supply the muscles with small amounts of ATP that are sufficient for quick bursts of activity. It also helps recycle NADH produced in glycolysis. When pyruvate is reduced to lactate via lactate dehydrogenase, the enzyme also reoxidizes NADH into NAD+, which is then funneled back into glycolysis in the oxidation of glucose to be used by other dehydrogenases. Even numbered fatty acids, the majority in the body, end up as two acetyl CoA molecules in the last step of β-oxidation in the breakdown of fatty acids. However, there are some odd numbered fatty acids as well, but they are not very common. They’re oxidation results in two different molecules – one acetyl CoA (2 carbons) and one propionyl CoA (3 carbons). This propionyl CoA does not have many functions by itself in the body, but because everything in biochemistry is salvaged, it can be converted to another molecule and be useful somewhere else. Through a carboxylation by propionyl CoA carboxylase, followed by an isomerization by a mutase, propionyl CoA is converted to succinyl CoA, a major component of the TCA cycle. This conversion is important because it allows for the utilization of a seemingly useless molecule, which eventually gets oxidized in the TCA cycle to liberate electrons and produce ATP. BONUS Question. 5pts. What is the most beautiful thing you learned about Biochemistry this semester?