HMB 105 Carbohydrate Metabolism Notes PDF

HMB 105: CARBOHYDRATE METABOLISM COURSE OUTLINE Purpose To deeply understand the carbohydrate metabolism (anabolism and catabolism) in mammalian cells. Course Content Lesson 1: Glycolytic Pathway; its regulation and Feeder pathways. Metabolic pathway (steps) of glycolysis in cells, its regulation a...

HMB 105: CARBOHYDRATE METABOLISM COURSE OUTLINE Purpose To deeply understand the carbohydrate metabolism (anabolism and catabolism) in mammalian cells. Course Content Lesson 1: Glycolytic Pathway; its regulation and Feeder pathways. Metabolic pathway (steps) of glycolysis in cells, its regulation and its feeder pathways. Lesson 2: Respiration; Kreb’s cycle and its regulation, Mitochondria shuttle system, Electron Transport Chain (ETC) and oxidation phosphorylation. The various pathways that lead to the generation of energy in form of ATP Lesson 3: Gluconeogenesis, and Coordinated Regulation of Glycolysis and Gluconeogenesis Metabolic pathway (steps) of gluconeogenesis in cells and its regulation. Lesson 4: Glyoxylate Cycle Metabolic pathway (steps) of the glyoxylate cycle its importance. Lesson 5: Hexose Monophosphate Pathway (HMP) Metabolic pathway (steps) of the Hexose Monophosphate Pathway (HMP) and its importance. Lesson 6: Glycogen metabolism and its regulation Metabolic pathway (steps) of glycogen synthesis (glycogenesis) and glycogen degradation (glycogenolysis), and their regulation. Mode of Delivery Lectures, discovery learning, problem-based learning, experiential learning, group-based learning, independent studies and e-learning. Assessment CATs and assignments 30%, End of semester Exam 70% Core Texts References a) Lehninger: Principles of Biochemistry (5th Edition) ISBN-13: 978-1464126116. David L. Nelson (Author), Michael M. Cox (Author) b) Weil, J.H (2005) General Biochemistry. New Age International Publishers Ltd. c) Campbell, K.M and Farrel, O.S (2012) Biochemistry (7th Ed.) Mary Finch. New York. d) Stryer, L., Tymoczko, J.L and Berg, M.J. (2005) Biochemistry. W.H Freeman and Company and Sumanas, Inc. 1 Lesson 1: Glycolytic Pathway; its Regulation and Feeder Pathways. 1.1 Introduction In this lesson, we introduce ourselves generally to carbohydrate metabolism i.e. glycolytic pathway and its regulation as well as the feeder pathways. Learning Outcomes By the end of this lesson you will be able to understand how: i. Glucose is broken down to generate energy. ii. How the glycolytic pathway is regulated. Glucose is the most abundant monosaccharide, the simplest form of carbohydrates, that is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using energy from sunlight. Glucose occupies a central position in the metabolism of plants, animals, and many microorganisms. It is relatively rich in potential energy, and thus a good fuel; the complete oxidation of glucose to carbon dioxide and water proceeds with a standard free-energy change of -2,840 kJ/mol. By storing glucose as a high molecular weight polymer such as starch or glycogen, a cell can stockpile large quantities of hexose units while maintaining a relatively low cytosolic osmolarity. When energy demands increase, glucose can be released from these intracellular storage polymers and used to produce ATP either aerobically or anaerobically. Glucose is also a remarkably versatile precursor, capable of supplying a huge array of metabolic intermediates for biosynthetic reactions; for example, a bacterium such as Escherichia coli can obtain from glucose the carbon skeletons for every amino acid, nucleotide, coenzyme, fatty acid, or other metabolic intermediate it needs for growth. In animals and vascular plants, glucose has four major fates: it may be used in the synthesis of complex polysaccharides destined for the extracellular space; stored in cells (as a polysaccharide or as sucrose); oxidized to a three-carbon compound (pyruvate) via glycolysis to provide ATP and metabolic intermediates; or oxidized via the pentose phosphate (phosphogluconate) pathway to yield ribose-5- phosphate for nucleic acid synthesis and NADPH for reductive biosynthetic processes (Fig. 14–1). 2 1.2 Glycolysis In glycolysis (from the Greek glykys, “sweet” or “sugar,” and lysis, “splitting”), the first metabolic pathway to be elucidated and is probably the best understood, a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis, some of the free energy released from glucose is conserved in the form of ATP and NADH. Glycolysis is an almost universal central pathway of glucose catabolism, the pathway with the largest flux of carbon in most cells. The glycolytic breakdown of glucose is the sole source of metabolic energy in some mammalian tissues and cell types (erythrocytes, renal medulla, brain, and sperm, for example). Some plant tissues that are modified to store starch (such as potato tubers) and some aquatic plants (watercress, for example) derive most of their energy from glycolysis; many anaerobic microorganisms are entirely dependent on glycolysis. Fermentation is a general term for the anaerobic degradation of glucose or other organic nutrients to obtain energy, conserved as ATP. Because living organisms first arose in an atmosphere without oxygen, anaerobic breakdown of glucose is probably the most ancient biological mechanism for obtaining energy from organic fuel molecules. And as genome sequencing of a wide variety of organisms has revealed, some archaea and some parasitic microorganisms lack one or more of the enzymes of glycolysis but retain the core of the pathway; they presumably carry out variant forms of glycolysis. In the course of evolution, the chemistry of this reaction sequence has been completely conserved; the glycolytic enzymes of vertebrates are closely similar, in amino acid sequence and three-dimensional structure, to their homologs in yeast and spinach. Glycolysis differs among species only in the details 3 of its regulation and in the subsequent metabolic fate of the pyruvate formed. The thermodynamic principles and the types of regulatory mechanisms that govern glycolysis are common to all pathways of cell metabolism. 1.2.1 The Two Phases of Glycolysis The breakdown of the six-carbon glucose into two molecules of the three-carbon pyruvate occurs in 10 steps, the first 5 of which constitute the preparatory phase (Fig. 14–2a). In these reactions, glucose is first phosphorylated at the hydroxyl group on C-6 (step 1). The D-glucose 6-phosphate thus formed is converted to D-fructose 6-phosphate (step 2), which is again phosphorylated, this time at C-1, to yield D-fructose-1,6-bisphosphate (step 3). For both phosphorylations, ATP is the phosphoryl group donor. As all sugar derivatives in glycolysis are the D isomers. Fructose 1,6-bisphosphate is split to yield two three-carbon molecules, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP) (step 4); this is the “lysis” step that gives the pathway its name. The dihydroxyacetone phosphate is isomerized to a second molecule of glyceraldehyde 3- phosphate (step 5), ending the first phase of glycolysis. From a chemical perspective, the isomerization in step 2 is critical for setting up the phosphorylation and C⎯C bond cleavage reactions in steps 3 and 4. Note that two molecules of ATP are invested before the cleavage of glucose into two three-carbon pieces; there will be a good return on this investment. To summarize: in the preparatory phase of glycolysis the energy of ATP is invested, raising the free energy content of the intermediates, and the carbon chains of all the metabolized hexoses are converted to a common product, glyceraldehyde 3-phosphate. The energy gain comes in the payoff phase of glycolysis (Fig. 14–2b). Each molecule of glyceraldehyde-3-phosphate is oxidized and phosphorylated by inorganic phosphate (not by ATP) to form 1,3-bisphosphoglycerate (step 6). Energy is then released as the two molecules of 1,3- bisphosphoglycerate are converted to two molecules of pyruvate (steps 7 through 10). Much of this energy is conserved by the coupled phosphorylation of four molecules of ADP to ATP. The net yield is two molecules of ATP per molecule of glucose used, because two molecules of ATP were invested in the preparatory phase. Energy is also conserved in the payoff phase in the formation of two molecules of the electron carrier NADH per molecule of glucose. In the sequential reactions of glycolysis, three types of chemical transformations are particularly noteworthy: (1) degradation of the carbon skeleton of glucose to yield pyruvate; (2) phosphorylation of ADP to ATP by compounds with high phosphoryl group transfer potential, formed during glycolysis; and (3) transfer of a hydride ion to NAD+, forming NADH. 4 Figure 14–2: The two phases of glycolysis. For each molecule of glucose that passes through the preparatory phase (a), two molecules of glyceraldehyde 3-phosphate are formed; both pass through the payoff phase (b). Pyruvate is the end product of the second phase of glycolysis. For each glucose molecule, two ATP are consumed in the preparatory phase and four ATP are produced in the payoff phase, giving a net yield of two ATP per molecule of glucose converted to pyruvate. The numbered 5 reaction steps are catalyzed by the enzymes listed on the right. Please note that each phosphoryl group, represented here as P, has two negative charges (---PO2-3). During glycolysis some of the energy of the glucose molecule is conserved in ATP, while much remains in the product, pyruvate. The overall equation for glycolysis is; NOTE: Glycolysis releases only a small fraction of the total available energy of the glucose molecule; the two molecules of pyruvate formed by glycolysis still contain most of the chemical potential energy of glucose, energy that can be extracted by oxidative reactions in the citric acid cycle and oxidative phosphorylation. 1.3 Fates of Pyruvate When oxygen is available, aerobic organisms completely oxidize pyruvate to CO2 and H2O via Acetyl- coA. In the absence of oxygen, pyruvate can be converted to several types of reduced molecules. In some cells (e.g., yeast), ethanol and CO2 are produced. In others (e.g., muscle cells), homolactic fermentation occurs in which lactate is the only organic product. Some microorganisms use heterolactic fermentation reactions that produce other acids or alcohols in addition to lactate. In all fermentation processes, the principal purpose is to regenerate NAD+ so that glycolysis can continue. 6 1. Pyruvate is oxidized, with loss of its carboxyl group as CO2, to yield the acetyl group of acetyl- coenzyme A; the acetyl group is then oxidized completely to CO2 by the citric acid cycle. The electrons from these oxidations are passed to O2 through a chain of carriers in mitochondria, to form H2O. The energy from the electron-transfer reactions drives the synthesis of ATP in mitochondria. Pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). 2. The second route for pyruvate is its reduction to lactate via lactic acid fermentation. When vigorously contracting skeletal muscle must function under low oxygen conditions (hypoxia), NADH cannot be reoxidized to NAD+, but NAD+ is required as an electron acceptor for the further oxidation of pyruvate. Under these conditions’ pyruvate is reduced to lactate, accepting electrons from NADH and thereby regenerating the NAD+ necessary for glycolysis to continue. Certain tissues and cell types (retina and erythrocytes, for example) convert glucose to lactate even under aerobic conditions, and lactate is also the product of glycolysis under anaerobic conditions in some microorganisms. 3. The third major route of pyruvate catabolism leads to ethanol. In some plant tissues and in certain invertebrates, protists, and microorganisms such as brewer’s or baker’s yeast, pyruvate is converted under hypoxic or anaerobic conditions to ethanol and CO2, a process called ethanol (alcohol) fermentation. 7 1.4 Regulation of Glycolysis The ATP yield from glycolysis under anaerobic conditions (2 ATP per molecule of glucose) is much smaller than that from the complete oxidation of glucose to CO2 under aerobic conditions (30 or 32 ATP per glucose). About 15 times as much glucose must therefore be consumed anaerobically as aerobically to yield the same amount of ATP. The flux of glucose through the glycolytic pathway is regulated to maintain nearly constant ATP levels (as well as adequate supplies of glycolytic intermediates that serve biosynthetic roles). The required adjustment in the rate of glycolysis is achieved by a complex interplay among; ATP consumption, NADH regeneration, and allosteric regulation of several glycolytic enzymes—including hexokinase, PFK-1, and pyruvate kinase—and by second-to-second fluctuations in the concentration of key metabolites that reflect the cellular balance between ATP production and consumption. On a slightly longer time scale, glycolysis is regulated by; the hormones glucagon, epinephrine, and insulin, and by changes in the expression of the genes for several glycolytic enzymes. An especially interesting case of abnormal regulation of glycolysis is seen in cancer. 1.5 Feeder Pathways for Glycolysis Many carbohydrates besides glucose meet their catabolic fate in glycolysis, after being transformed into one of the glycolytic intermediates. The most significant are the storage polysaccharides glycogen and starch, either within cells (endogenous) or obtained in the diet; the disaccharides maltose, lactose, trehalose, and sucrose; and the monosaccharides fructose, mannose, and galactose (Fig. 14–10). 8 1.5.1 Dietary Polysaccharides and Disaccharides Undergo Hydrolysis to Monosaccharides For most humans, starch is the major source of carbohydrates in the diet (Fig. 14–10). Digestion begins in the mouth, where salivary a-amylase hydrolyzes the internal (a1—4) glycosidic linkages of starch, producing short polysaccharide fragments or oligosaccharides. (Note that in this hydrolysis reaction, water, not Pi, is the attacking species.) In the stomach, salivary a-amylase is inactivated by the low pH, but a second form of a-amylase, secreted by the pancreas into the small intestine, continues the breakdown process. Pancreatic a- amylase yields mainly maltose and maltotriose (the di- and trisaccharides of glucose) and oligosaccharides called limit dextrins, fragments of amylopectin containing (a1—6) branch points. Maltose and dextrins are degraded to glucose by enzymes of the intestinal brush border (the fingerlike microvilli of intestinal epithelial cells, which greatly increase the area of the intestinal surface). Dietary glycogen has essentially the same structure as starch, and its digestion proceeds by the same pathway. 9 1.5.2 Endogenous Glycogen and Starch Degradation (Phosphorolysis) Glycogen stored in animal tissues (primarily liver and skeletal muscle), in microorganisms, or in plant tissues can be mobilized for use within the same cell by a phosphorolytic reaction catalyzed by glycogen phosphorylase (starch phosphorylase in plants) (Fig. 14–11). These enzymes catalyze an attack by Pi on the (a1—4) glycosidic linkage that joins the last two glucose residues at a nonreducing end, generating glucose-1-phosphate and a polymer one glucose unit shorter. Phosphorolysis preserves some of the energy of the glycosidic bond in the phosphate ester glucose- 1-phosphate. Glycogen phosphorylase (or starch phosphorylase) acts repetitively until it approaches an (a1—6) branch point, where its action stops. A debranching enzyme removes the branches. The mechanisms and control of glycogen degradation are described in greater detail later. Glucose-1-phosphate produced by glycogen phosphorylase is converted to glucose-6-phosphate by phosphoglucomutase, which catalyzes the reversible reaction Phosphoglucomutase employs essentially the same mechanism as phosphoglycerate mutase: both entail a bisphosphate intermediate, and the enzyme is transiently phosphorylated in each catalytic 10 cycle. The glucose 6-phosphate formed in the phosphoglucomutase reaction can enter glycolysis or another pathway such as the pentose phosphate pathway, described later in this module. NOTE: Breakdown of dietary polysaccharides such as glycogen and starch in the gastrointestinal tract by phosphorolysis rather than hydrolysis would produce no energy gain: sugar phosphates are not transported into the cells that line the intestine, but must first be dephosphorylated to the free sugar. Disaccharides must be hydrolyzed to monosaccharides before entering cells. Intestinal disaccharides and dextrins are hydrolyzed by enzymes attached to the outer surface of the intestinal epithelial cells: The monosaccharides so formed are actively transported into the epithelial cells, then passed into the blood to be carried to various tissues, where they are phosphorylated and funneled into the glycolytic sequence. HEALTH BYTE: Lactose intolerance, common among adults of most human populations except those originating in Northern Europe and some parts of Africa, is due to the disappearance after childhood of most or all of the lactase activity of the intestinal epithelial cells. Without intestinal lactase, lactose cannot be completely digested and absorbed in the small intestine, and it passes into the large intestine, where bacteria convert it to toxic products that cause abdominal cramps and diarrhea. The problem is further complicated because undigested lactose and its metabolites increase the osmolarity of the intestinal contents, favoring retention of water in the intestine. In most parts of the world where lactose intolerance is prevalent, milk is not used as a food by adults, although milk products pre-digested with lactase are commercially available in some countries. 1.5.3 Other Monosaccharides Enter the Glycolytic Pathway at Several Points 1.5.3.1 D-Fructose In most organisms, hexoses other than glucose can undergo glycolysis after conversion to a phosphorylated derivative. D-Fructose, present in free form in many fruits and formed by hydrolysis of sucrose in the small intestine of vertebrates, is phosphorylated by hexokinase: 11 This is a major pathway of fructose entry into glycolysis in the muscles and kidney. In the liver, fructose enters by a different pathway. The liver enzyme fructokinase catalyzes the phosphorylation of fructose at C-1 rather than C-6: The fructose-1-phosphate is then cleaved to glyceraldehyde and dihydroxyacetone phosphate by fructose-1-phosphate aldolase: Dihydroxyacetone phosphate is converted to glyceraldehyde-3-phosphate by the glycolytic enzyme triose phosphate isomerase. Glyceraldehyde is phosphorylated by ATP and triose kinase to glyceraldehyde-3-phosphate: Thus, both products of fructose-1-phosphate hydrolysis enter the glycolytic pathway as glyceraldehyde-3-phosphate. 1.5.3.2 D-Galactose D-Galactose, a product of the hydrolysis of lactose (milk sugar), passes in the blood from the intestine to the liver, where it is first phosphorylated at C-1, at the expense of ATP, by the enzyme galactokinase: The galactose-1-phosphate is then converted to its epimer at C-4, glucose-1-phosphate, by a set of reactions in which uridine diphosphate (UDP) functions as a coenzyme-like carrier of hexose groups as shown in (Fig. 14–12) below. 12 The epimerization involves first the oxidation of the C-4 —OH group to a ketone, then reduction of the ketone to an —OH, with inversion of the configuration at C-4. NAD is the cofactor for both the oxidation and the reduction. HEALTH BYTE: A defect in any of the three enzymes in this pathway causes galactosemia in humans. In galactokinase deficiency galactosemia, high galactose concentrations are found in blood and urine. Affected individuals develop cataracts in infancy, caused by deposition of the galactose metabolite galactitol in the lens. The other symptoms in this disorder are relatively mild, and strict limitation of galactose in the diet greatly diminishes their severity. Transferase-deficiency 13 galactosemia is more serious; it is characterized by poor growth in childhood, speech abnormality, mental deficiency, and liver damage that may be fatal, even when galactose is withheld from the diet. Epimerase-deficiency galactosemia leads to similar symptoms, but is less severe when dietary galactose is carefully controlled. 1.5.3.3 D-Mannose D-Mannose, released in the digestion of various polysaccharides and glycoproteins of foods, can be phosphorylated at C-6 by hexokinase: Mannose 6-phosphate is isomerized by phosphomannose isomerase to yield fructose 6-phosphate, an intermediate of glycolysis. 14 Numbering, Lesson 1 pacing and sequencing Title Glycolytic Pathway; its regulation and feeder pathways. Purpose To generally introduce you to carbohydrate metabolism ; the various pathways that lead to the generation of energy; Glycolysis. Brief summary of Please watch: Glycolysis: https://youtu.be/C5wNfdWr4tk overall task Spark Individual Watch the video contribution On the discussion forum, state the key steps in the regulation of glycolysis and its role in the food and beverage industry Interaction begins Post your answers on the discussion forum Look at your classmate’s responses. E-moderator Providing feedback/ teaching points interventions Summarising key points Closing the discussion Schedule and time This discussion should be done between 7th September, 2020 and 11th September, 2020 Next Kreb’s Cycle and its regulation, Mitochondria shuttle system, and electron transport chain and oxidation phosphorylation. 15 2. Lesson 2: Citric acid cycle (TCA) and its regulation; Electron Transport Chain (ETC) and oxidation phosphorylation 2.1 Introduction The citric acid cycle is also called the tricarboxylic acid (TCA) cycle or the Krebs cycle (after its discoverer, Hans Krebs). Some cells obtain energy (ATP) by fermentation, breaking down glucose in the absence of oxygen. For most eukaryotic cells and many bacteria, which live under aerobic conditions and oxidize their organic fuels to carbon dioxide and water, glycolysis is but the first stage in the complete oxidation of glucose. Rather than being reduced to lactate, ethanol, or some other fermentation product, the pyruvate produced by glycolysis is further oxidized to H2O and CO2. This aerobic phase of catabolism is called respiration. In the broader physiological or macroscopic sense, respiration refers to a multicellular organism’s uptake of O2 and release of CO2. Biochemists and cell biologists, however, use the term in a narrower sense to refer to the molecular processes by which cells consume O2 and produce CO2—processes more precisely termed cellular respiration. Cellular respiration occurs in three major stages as shown in (Fig. 16–1): 1. In the first, organic fuel molecules—glucose, fatty acids, and some amino acids—are oxidized to yield two-carbon fragments in the form of the acetyl group of acetyl-coenzyme A (acetyl- CoA). 2. In the second stage, the acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them to CO2; the energy released is conserved in the reduced electron carriers; NADH and FADH2. 3. In the third stage of respiration, these reduced coenzymes are themselves oxidized, giving up protons (H+) and electrons. The electrons are transferred to O2—the final electron acceptor—via a chain of electron-carrying molecules known as the respiratory chain. In the course of electron transfer, the large amount of energy released is conserved in the form of ATP, by a process called oxidative phosphorylation that will be discussed later. Respiration is more complex than glycolysis and is believed to have evolved much later, after the appearance of cyanobacteria. 16 2.2 Production of Acetyl-CoA (Activated Acetate) In aerobic organisms, glucose and other sugars, fatty acids, and most amino acids are ultimately oxidized to CO2 and H2O via the citric acid cycle and the respiratory chain. Before entering the citric acid cycle, the carbon skeletons of sugars and fatty acids are degraded to the acetyl group of acetyl- CoA, the form in which the cycle accepts most of its fuel input. Many amino acid carbons also enter the cycle this way, although several amino acids are degraded to other cycle intermediates. In this lesson, we focus on how pyruvate, derived from glucose and other sugars by glycolysis, is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex, a cluster of 17 enzymes—multiple copies of each of three enzymes—located in the mitochondria of eukaryotic cells and in the cytosol of bacteria. The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA as shown below in (Fig. 16–2). The NADH formed in this reaction gives up a hydride ion (:H-) to the respiratory chain (Fig. 16–1), which carries the two electrons to oxygen or, in anaerobic microorganisms, to an alternative electron acceptor such as nitrate or sulfate. The transfer of electrons from NADH to oxygen ultimately generates 2.5 molecules of ATP per pair of electrons. The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA as shown above requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups. The PDH complex contains three enzymes; a) Pyruvate dehydrogenase (E1) b) Dihydrolipoyl transacetylase (E2) c) Dihydrolipoyl dehydrogenase (E3) Each enzyme is present in multiple copies. The number of copies of each enzyme and therefore the size of the complex varies among species. The five different coenzymes or prosthetic groups include; a) Thiamine pyrophosphate (TPP) b) Flavin adenine dinucleotide (FAD) c) Coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group) d) nicotinamide adenine dinucleotide (NAD) e) Lipoate. 18  HEALTH BYTE: Four different vitamins required in human nutrition are vital components of this system: thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already stated the roles of FAD and NAD as electron carriers. Consequently, mutations in the genes for the subunits of the PDH complex, or a dietary thiamine deficiency, can have severe consequences. Thiamine-deficient animals are unable to oxidize pyruvate normally. This is of particular importance to the brain, which usually obtains all its energy from the aerobic oxidation of glucose in a pathway that necessarily includes the oxidation of pyruvate. Beriberi, a disease that results from thiamine deficiency, is characterized by loss of neural function. This disease occurs primarily in populations that rely on a diet consisting mainly of white (polished) rice, which lacks the hulls in which most of the thiamine of rice is found. People who habitually consume large amounts of alcohol can also develop thiamine deficiency, because much of their dietary intake consists of the vitamin-free “empty calories” of distilled spirits. An elevated level of pyruvate in the blood is often an indicator of defects in pyruvate oxidation due to one of these causes. 2.3 Reactions of the Citric Acid Cycle The process by which acetyl-CoA undergoes oxidation, the citric acid cycle, is a cyclic pathway as shown in the figure below (Fig. 16–7). The citric acid cycle has eight steps that takes place in the mitochondria. Mitochondria also contain all the enzymes and proteins necessary for the last stage of respiration—electron transfer and ATP synthesis by oxidative phosphorylation. Besides, mitochondria also contain the enzymes for the oxidation of fatty acids and some amino acids to acetyl- CoA, and the oxidative degradation of other amino acids to a -ketoglutarate, succinyl-CoA, or oxaloacetate. Thus, in nonphotosynthetic eukaryotes, the mitochondrion is the site of most energy-yielding oxidative reactions and of the coupled synthesis of ATP. In photosynthetic eukaryotes, mitochondria are the major site of ATP production in the dark, but in daylight chloroplasts produce most of the organism’s ATP. In most bacteria, the enzymes of the citric acid cycle are in the cytosol, and the plasma membrane plays a role analogous to that of the inner mitochondrial membrane in ATP synthesis. To begin a turn of the cycle, acetyl-CoA donates its acetyl group to the four-carbon compound oxaloacetate to form the six-carbon citrate. Citrate is then transformed into isocitrate, also a six- carbon molecule, which is dehydrogenated with loss of CO2 to yield the five-carbon compound a- ketoglutarate (also called oxoglutarate). a-Ketoglutarate undergoes loss of a second molecule of CO2 and ultimately yields the four-carbon compound succinate. Succinate is then enzymatically 19 converted in three steps into the four-carbon oxaloacetate—which is then ready to react with another molecule of acetyl-CoA. In each turn of the cycle, one acetyl group (two carbons) enters as acetyl-CoA and two molecules of CO2 leave; one molecule of oxaloacetate is used to form citrate and one molecule of oxaloacetate is regenerated. No net removal of oxaloacetate occurs; one molecule of oxaloacetate can theoretically bring about oxidation of an infinite number of acetyl groups, and, in fact, oxaloacetate is present in cells in very low concentrations. Four of the eight steps in this process are oxidations, in which the energy of oxidation is very efficiently conserved in the form of the reduced coenzymes NADH and FADH2. Although the citric acid cycle is central to energy-yielding metabolism its role is not limited to energy conservation. Four- and five-carbon intermediates of the cycle serve as precursors for a wide variety of products. To replace intermediates removed for this purpose, cells employ anaplerotic (replenishing) reactions. In oxidative phosphorylation, passage of two electrons from NADH to O2 drives the formation of about 2.5 ATP, and passage of two electrons from FADH2 to O2 yields about 1.5 ATP. This stoichiometry allows us to calculate the overall yield of ATP from the complete oxidation of glucose. When both pyruvate molecules are oxidized to 6 CO2 via the pyruvate dehydrogenase complex and the citric acid cycle, and the electrons are transferred to O2 via oxidative phosphorylation, as many as 32 ATP are obtained per glucose (Table 16–1). In round numbers, this represents the conservation of 32 x 30.5 kJ/mol = 976 kJ/mol, or 34% of the theoretical maximum of about 2,840 kJ/mol available from the complete oxidation of glucose. These calculations employ the standard free-energy changes; when corrected for the actual free energy required to form ATP within cells, the calculated efficiency of the process is closer to 65%. 20 21 NOTE: The citric acid cycle components are important biosynthetic intermediates. In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes. Besides its role in the oxidative catabolism of carbohydrates, fatty acids, and amino acids, the cycle provides precursors for many biosynthetic pathways as shown in (Fig. 16–15). 22 As intermediates of the citric acid cycle are removed to serve as biosynthetic precursors, they are replenished by anaplerotic reactions (Fig. 16–15; Table 16–2). Under normal circumstances, the reactions by which cycle intermediates are siphoned off into other pathways and those by which they are replenished are in dynamic balance, so that the concentrations of the citric acid cycle intermediates remain almost constant. 23 2.4 Regulation of the Citric Acid Cycle The regulation of key enzymes in metabolic pathways, by allosteric effectors and by covalent modification, ensures the production of intermediates at the rates required to keep the cell in a stable steady state while avoiding wasteful overproduction. The flow of carbon atoms from pyruvate into and through the citric acid cycle is under tight regulation at two levels: a) The conversion of pyruvate to acetyl-CoA, the starting material for the cycle (the pyruvate dehydrogenase complex reaction). b) The entry of acetyl-CoA into the cycle (the citrate synthase reaction). Acetyl-CoA is also produced by pathways other than the PDH complex reaction—most cells produce acetyl-CoA from the oxidation of fatty acids and certain amino acids—and the availability of intermediates from these other pathways is important in the regulation of pyruvate oxidation and of the citric acid cycle. The cycle is also regulated at the isocitrate dehydrogenase and a-ketoglutarate dehydrogenase reactions. Production of acetyl-CoA by the pyruvate dehydrogenase complex is regulated by allosteric and covalent mechanisms. The PDH complex of mammals is strongly inhibited by ATP and by acetyl- CoA and NADH, the products of the reaction catalyzed by the complex as shown in (Fig. 16–18). The allosteric inhibition of pyruvate oxidation is greatly enhanced when long-chain fatty acids are available. AMP, CoA, and NAD+, all of which accumulate when too little acetate flows into the citric acid cycle, allosterically activate the PDH complex. Thus, this enzyme activity is turned off when ample fuel is available in the form of fatty acids and acetyl-CoA and when the cell’s [ATP]/[ADP] and [NADH]/[NAD+] ratios are high, and it is turned on again when energy demands are high and the cell requires greater flux of acetyl-CoA into the citric acid cycle. In mammals, these allosteric regulatory mechanisms are complemented by a second level of regulation: covalent protein modification. The PDH complex is inhibited by reversible phosphorylation of a specific Ser residue on one of the two subunits of E1. In addition to the enzymes E1, E2, and E3, the mammalian PDH complex contains two regulatory proteins whose sole purpose is to regulate the activity of the complex. A specific protein kinase phosphorylates and thereby inactivates E1, and a specific phosphoprotein phosphatase removes the phosphoryl group by hydrolysis and thereby activates E1. The kinase is allosterically activated by ATP: when [ATP] is high (reflecting a sufficient supply of energy), the PDH complex is inactivated by phosphorylation of E1. When [ATP] declines, kinase activity decreases and phosphatase action removes the phosphoryl groups from E1, activating the complex. The PDH complex of plants, located in the mitochondrial matrix and in 24 plastids, is inhibited by its products, NADH and acetyl-CoA. The plant mitochondrial enzyme is also regulated by reversible phosphorylation; pyruvate inhibits the kinase, thus activating the PDH complex, and NH+4 stimulates the kinase, causing inactivation of the complex. The PDH complex of E. coli is under allosteric regulation similar to that of the mammalian enzyme, but it does not seem to be regulated by phosphorylation. The flow of metabolites through the citric acid cycle is under stringent regulation. Three factors govern the rate of flux through the cycle: a) substrate availability b) inhibition by accumulating products, and c) allosteric feedback inhibition of the enzymes that catalyze early steps in the cycle. Each of the three strongly exergonic steps in the cycle—those catalyzed by citrate synthase, isocitrate 25 dehydrogenase, and a-ketoglutarate dehydrogenase (Fig. 16–18)—can become the rate-limiting step under some circumstances. The availability of the substrates for citrate synthase (acetyl-CoA and oxaloacetate) varies with the metabolic state of the cell and sometimes limits the rate of citrate formation. NADH, a product of isocitrate and a-ketoglutarate oxidation, accumulates under some conditions, and at high [NADH]/[NAD+] both dehydrogenase reactions are severely inhibited by mass action. Similarly, in the cell, the malate dehydrogenase reaction is essentially at equilibrium (that is, it is substrate-limited), and when [NADH]/[NAD+] is high the concentration of oxaloacetate is low, slowing the first step in the cycle. Product accumulation inhibits all three limiting steps of the cycle: succinyl-CoA inhibits a- ketoglutarate dehydrogenase (and also citrate synthase); citrate blocks citrate synthase; and the end product, ATP, inhibits both citrate synthase and isocitrate dehydrogenase. The inhibition of citrate synthase by ATP is relieved by ADP, an allosteric activator of this enzyme. In vertebrate muscle, Ca2+, the signal for contraction and for a concomitant increase in demand for ATP, activates both isocitrate dehydrogenase and a-ketoglutarate dehydrogenase, as well as the PDH complex. In short, the concentrations of substrates and intermediates in the citric acid cycle set the flux through this pathway at a rate that provides optimal concentrations of ATP and NADH. Under normal conditions, the rates of glycolysis and of the citric acid cycle are integrated so that only as much glucose is metabolized to pyruvate as is needed to supply the citric acid cycle with its fuel, the acetyl groups of acetyl-CoA. Pyruvate, lactate, and acetyl-CoA are normally maintained at steady-state concentrations. The rate of glycolysis is matched to the rate of the citric acid cycle not only through its inhibition by high levels of ATP and NADH, which are common to both the glycolytic and respiratory stages of glucose oxidation, but also by the concentration of citrate. Citrate, the product of the first step of the citric acid cycle, is an important allosteric inhibitor of phosphofructokinase-1 in the glycolytic pathway (see Fig. 15–14). HEALTH BYTE: Some mutations in enzymes of the citric acid cycle may lead to cancer. When the mechanisms for regulating a pathway such as the citric acid cycle are overwhelmed by a major metabolic perturbation, the result can be serious disease. Mutations in citric acid cycle enzymes are very rare in humans and other mammals, but those that do occur are devastating. Genetic defects in the fumarase gene lead to tumors of smooth muscle (leiomas) and kidney; mutations in succinate 26 dehydrogenase lead to tumors of the adrenal gland (pheochromocytomas). In cultured cells with these mutations, fumarate (in the case of fumarase mutations) and, to a lesser extent, succinate (in the case of succinate dehydrogenase mutations) accumulate, and this accumulation induces the hypoxia- inducible transcription factor HIF-1a. The mechanism of tumor formation may be the production of a pseudohypoxic state. In cells with these mutations, there is an up-regulation of genes normally regulated by HIF-1a. These effects of mutations in the fumarase and succinate dehydrogenase genes define them as tumor suppressor genes. 2.5 Mitochondria Shuttle System. Shuttle systems indirectly convey cytosolic NADH into mitochondria for oxidation. 2.5.1 Malate-Aspartate Shuttle The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix. Given that the inner membrane is not permeable to NADH, how can the NADH generated by glycolysis in the cytosol be reoxidized to NAD+ by O2 via the respiratory chain? Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route. The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle as illustrated in (Fig. 19–29). 27 The reducing equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to yield malate, catalyzed by cytosolic malate dehydrogenase. The malate thus formed passes through the inner membrane via the malate–a-ketoglutarate transporter. Within the matrix the reducing equivalents are passed to NAD+ by the action of matrix malate dehydrogenase, forming NADH; this NADH can pass electrons directly to the respiratory chain. About 2.5 molecules of ATP are generated as this pair of electrons passes to O2. Cytosolic oxaloacetate must be regenerated by transamination reactions and the activity of membrane transporters to start another cycle of the shuttle. 2.5.2 Glycerol 3-phosphate Shuttle Skeletal muscle and brain use a different NADH shuttle, the glycerol 3-phosphate shuttle (Fig. 19– 30). It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH to ubiquinone and thus into Complex III, not Complex I, providing only enough energy to synthesize 1.5 ATP molecules per pair of electrons. The mitochondria of plants have an externally oriented NADH dehydrogenase that can transfer electrons directly from cytosolic NADH into the respiratory chain at the level of ubiquinone. Because this pathway bypasses the NADH dehydrogenase of Complex I and the associated proton movement, the yield of ATP from cytosolic NADH is less than that from NADH generated in the matrix. 28 2.6 Oxidative phosphorylation Oxidative phosphorylation is the culmination of energy-yielding metabolism in aerobic organisms. All oxidative steps in the degradation of carbohydrates, fats, and amino acids converge at this final stage of cellular respiration, in which the energy of oxidation drives the synthesis of ATP. Photophosphorylation is the means by which photosynthetic organisms capture the energy of sunlight—the ultimate source of energy in the biosphere—and harness it to make ATP. Together, oxidative phosphorylation and photophosphorylation account for most of the ATP synthesized by most organisms most of the time. In eukaryotes, oxidative phosphorylation occurs in mitochondria, photophosphorylation in chloroplasts. Oxidative phosphorylation involves the reduction of O2 to H2O with electrons donated by NADH and FADH2; it occurs equally well in light or darkness. Photophosphorylation involves the oxidation of H2O to O2, with NADP+ as ultimate electron acceptor; it is absolutely dependent on the energy of light. Despite their differences, these two highly efficient energy-converting processes have fundamentally similar mechanisms. Our current understanding of ATP synthesis in mitochondria and chloroplasts is based on the hypothesis, introduced by Peter Mitchell in 1961, that transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation reactions. This chemiosmotic theory has been accepted as one of the great unifying principles of twentieth century biology. It provides insight into the processes of oxidative phosphorylation and photophosphorylation, and into such apparently disparate energy transductions as active transport across membranes and the motion of bacterial flagella. Oxidative phosphorylation and photophosphorylation are mechanistically similar in three respects. a) Both processes involve the flow of electrons through a chain of membrane-bound carriers. b) The free energy made available by this “downhill” (exergonic) electron flow is coupled to the “uphill” transport of protons across a proton-impermeable membrane, conserving the free energy of fuel oxidation as a transmembrane electrochemical potential. c) The transmembrane flow of protons down their concentration gradient through specific protein channels provides the free energy for synthesis of ATP, catalyzed by a membrane protein complex (ATP synthase) that couples proton flow to phosphorylation of ADP. 29 2.6.1 Electron-Transfer Reactions in Mitochondria The mitochondrial respiratory chain consists of a series of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons. Three types of electron transfers occur in oxidative phosphorylation: (1) direct transfer of electrons, as in the reduction of Fe3+ to Fe2+; (2) transfer as a hydrogen atom (H+ + e-); and (3) transfer as a hydride ion (:H-), which bears two electrons. The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation-reduction reaction. In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain: a hydrophobic quinone (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron-sulfur proteins). Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble benzoquinone with a long isoprenoid side chain. The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron transfer chains. 30  a) Complex I: NADH to Ubiquinone Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase b) Complex II: Succinate to Ubiquinone Complex II, also called succinate dehydrogenase c) Complex III: Ubiquinone to Cytochrome c Complex III, also called cytochrome bc1 complex or ubiquinone:cytochrome c oxidoreductase d) Complex IV: Cytochrome c to O2 Complex IV, also called cytochrome oxidase 2.6.2 ATP Synthesis According to the chemiosmotic model, proposed by Peter Mitchell, the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane—the proton-motive force—drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with ATP synthase as illustrated in the figure below. 31 2.7 Regulation of Oxidative Phosphorylation Oxidative phosphorylation is regulated by cellular energy needs. The rate of respiration (O2 consumption) in mitochondria is tightly regulated; it is generally limited by the availability of ADP as a substrate for phosphorylation. Dependence of the rate of O2 consumption on the availability of the Pi acceptor ADP, the acceptor control of respiration, can be remarkable. In some animal tissues, the acceptor control ratio, the ratio of the maximal rate of ADP-induced O2 consumption to the basal rate in the absence of ADP, is at least 10. The intracellular concentration of ADP is one measure of the energy status of cells. Another, related measure is the mass-action ratio of the ATP-ADP system, [ATP]/([ADP][Pi]). Normally this ratio is very high, so the ATP-ADP system is almost fully phosphorylated. When the rate of some energy- requiring process (protein synthesis, for example) increases, the rate of breakdown of ATP to ADP and Pi increases, lowering the mass-action ratio. With more ADP available for oxidative phosphorylation, the rate of respiration increases, causing regeneration of ATP. This continues until the mass action ratio returns to its normal high level, at which point respiration slows again. The rate of oxidation of cellular fuels is regulated with such sensitivity and precision that the [ATP]/([ADP][Pi]) ratio fluctuates only slightly in most tissues, even during extreme variations in energy demand. In short, ATP is formed only as fast as it is used in energy-requiring cellular activities. Numbering, pacing Lesson 2 and sequencing 32 Title Citric acid cycle and its regulation; Mitochondria shuttle system, electron transport chain and oxidation phosphorylation Purpose To generally indulge deeper into carbohydrate metabolism (respiration); the various pathways that lead to the generation of energy in form of ATP; TCA, ETC & Oxidation Phosphorylation. Brief summary of Please watch: TCA Cycle; https://youtu.be/ubzw64PQPqM overall task Mitochondria Shuttle System; https://youtu.be/sS5ehPKUdac https://youtu.be/7RcmiSdQ3Yc ETC and Oxidation Phosphorylation; https://youtu.be/C8VHyezOJD4 https://youtu.be/E_UG3WnsW3o Spark Individual Watch the animation contribution On the discussion forum, state the key steps in the regulation of TCA, and discuss industrial applications of citrate. Interaction begins Post your answers on the discussion forum Look at your classmate’s responses. E-moderator Providing feedback/ teaching points interventions Summarising key points & closing the discussion Schedule and time This discussion should be done between 7th September, 2020 and 11th September, 2020 Next Gluconeogenesis, and Coordinated Regulation of Glycolysis and Gluconeogenesis 33 3. Lesson 3: Gluconeogenesis, and Coordinated Regulation of Glycolysis and Gluconeogenesis 3.1 Introduction Glucose plays a central role in metabolism and is nearly a universal fuel and building block in modern organisms, from microbes to humans. In mammals, some tissues depend almost completely on glucose for their metabolic energy. For the human brain and nervous system, as well as the erythrocytes, testes, renal medulla, and embryonic tissues, glucose from the blood is the sole or major fuel source. The brain alone requires about 120 g of glucose each day—more than half of all the glucose stored as glycogen in muscle and liver. However, the supply of glucose from these stores is not always sufficient; between meals and during longer fasts, or after vigorous exercise, glycogen is depleted. For these times, organisms need a method for synthesizing glucose from noncarbohydrate precursors. This is accomplished by a pathway called gluconeogenesis (“new formation of sugar”), which converts pyruvate and related three- and four-carbon compounds to glucose. Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms. The reactions are essentially the same in all tissues and all species. The important precursors of glucose in animals are three-carbon compounds such as lactate, pyruvate, and glycerol, as well as certain amino acids as shown in the (Fig. 14–15) below. In mammals, gluconeogenesis takes place mainly in the liver, and to a lesser extent in renal cortex and in the epithelial cells that line the inside of the small intestine. The glucose produced passes into the blood to supply other tissues. After vigorous exercise, lactate produced by anaerobic glycolysis in skeletal muscle returns to the liver and is converted to glucose, which moves back to muscle and is converted to glycogen—a circuit called the Cori cycle. In plant seedlings, stored fats and proteins are converted, via paths that include gluconeogenesis, to the disaccharide sucrose for transport throughout the developing plant. Glucose and its derivatives are precursors for the synthesis of plant cell walls, nucleotides and coenzymes, and a variety of other essential metabolites. In many microorganisms, gluconeogenesis starts from simple organic compounds of two or three carbons, such as acetate, lactate, and propionate, in their growth medium. 34 FIGURE 14–15: Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids. Plants and photosynthetic bacteria are uniquely able to convert CO2 to carbohydrates, using the glyoxylate cycle (discussed in the next lesson). Although the reactions of gluconeogenesis are the same in all organisms, the metabolic context and the regulation of the pathway differ from one species to another and from tissue to tissue. In this lesson we focus on gluconeogenesis as it occurs in the mammalian liver. Gluconeogenesis and glycolysis are not identical pathways running in opposite directions, although they do share several steps (Fig. 14–16); 7 of the 10 enzymatic reactions of gluconeogenesis are the reverse of glycolytic reactions. However, three reactions of glycolysis are essentially irreversible in vivo and cannot be used in gluconeogenesis: 35  a) the conversion of glucose to glucose 6-phosphate by hexokinase, b) the phosphorylation of fructose 6-phosphate to fructose-1,6-bisphosphate by phosphofructokinase-1, c) and the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase. In cells, these three reactions are characterized by a large negative free-energy change, whereas other glycolytic reactions have a ∆G near 0. In gluconeogenesis, the three irreversible steps are bypassed by a separate set of enzymes, catalyzing reactions that are sufficiently exergonic to be effectively irreversible in the direction of glucose synthesis. Thus, both glycolysis and gluconeogenesis are irreversible processes in cells. In animals, both pathways occur largely in the cytosol, necessitating their reciprocal and coordinated regulation. Separate regulation of the two pathways is brought about through controls exerted on the enzymatic steps unique to each. Please note the three bypass reactions of gluconeogenesis. (Keep in mind that “bypass” refers throughout to the bypass of irreversible glycolytic reactions.) 36 FIGURE 14–16 Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here will be discussed later in this lesson. Figure 14–19 below illustrates an alternative route for oxaloacetate produced in mitochondria. 37 FIGURE 14–19: Alternative paths from pyruvate to phosphoenolpyruvate. The relative importance of the two pathways depends on the availability of lactate or pyruvate and the cytosolic requirements for NADH for gluconeogenesis. The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion. 3.2 Coordinated Regulation of Glycolysis and Gluconeogenesis In mammals, gluconeogenesis occurs primarily in the liver, where its role is to provide glucose for export to other tissues when glycogen stores are exhausted and when no dietary glucose is available. As discussed above, gluconeogenesis employs several of the enzymes that act in glycolysis, but it is not simply the reversal of glycolysis. Seven of the glycolytic reactions are freely reversible, and the enzymes that catalyze these reactions also function in gluconeogenesis. Three reactions of glycolysis 38 are so exergonic as to be essentially irreversible: those catalyzed by hexokinase, PFK-1, and pyruvate kinase. All three reactions have a large, negative ∆G’. Gluconeogenesis uses detours around each of these irreversible steps; for example, the conversion of fructose-1,6-bisphosphate to fructose-6- phosphate is catalyzed by fructose-1,6-bisphosphatase (FBPase-1). Each of these bypass reactions also has a large, negative ∆G’. At each of the three points where glycolytic reactions are bypassed by alternative, gluconeogenic reactions, simultaneous operation of both pathways would consume ATP without accomplishing any chemical or biological work. For example, PFK-1 and FBPase-1 catalyze opposing reactions: that is, hydrolysis of ATP without any useful metabolic work being done. Clearly, if these two reactions were allowed to proceed simultaneously at a high rate in the same cell, a large amount of chemical energy would be dissipated as heat. This uneconomical process has been called a futile cycle. However, such cycles may provide advantages for controlling pathways, and the term substrate cycle is a better description. Similar substrate cycles also occur with the other two sets of bypass reactions of gluconeogenesis. We look now in some detail at the mechanisms that regulate glycolysis and gluconeogenesis at the three points where these pathways diverge: 3.2.1 Hexokinase Isozymes The hexokinase of muscle and liver are affected differently by their product, glucose-6-phosphate. Hexokinase, which catalyzes the entry of glucose into the glycolytic pathway, is a regulatory enzyme. Humans have four isozymes (designated I to IV), encoded by four different genes. Isozymes are different proteins that catalyze the same reaction. The predominant hexokinase isozyme of myocytes (hexokinase II) has a high affinity for glucose—it is half-saturated at about 0.1 mM. Because glucose entering myocytes from the blood (where the glucose concentration is 4 to 5 mM) produces an intracellular glucose concentration high enough to saturate hexokinase II, the enzyme normally acts at or near its maximal rate. Muscle hexokinase I and hexokinase II are allosterically inhibited by their product, glucose 6-phosphate, so whenever the cellular concentration of glucose-6-phosphate rises above its normal level, these isozymes are temporarily and reversibly inhibited, bringing the rate of glucose-6-phosphate formation into balance with the rate of its utilization and re-establishing the 39 steady state. The different hexokinase isozymes of liver and muscle reflect the different roles of these organs in carbohydrate metabolism: muscle consumes glucose, using it for energy production, whereas liver maintains blood glucose homeostasis by consuming or producing glucose, depending on the prevailing blood glucose concentration. The predominant hexokinase isozyme of liver is hexokinase IV (glucokinase), which differs in three important respects from hexokinases I–III of muscle. First, the glucose concentration at which hexokinase IV is half-saturated (about 10 mM) is higher than the usual concentration of glucose in the blood. Because an efficient glucose transporter in hepatocytes (GLUT2) rapidly equilibrates the glucose concentrations in cytosol and blood, the high Km of hexokinase IV allows its direct regulation by the level of blood glucose. When blood glucose is high, as it is after a meal rich in carbohydrates, excess glucose is transported into hepatocytes, where hexokinase IV converts it to glucose 6- phosphate. Because hexokinase IV is not saturated at 10 mM glucose, its activity continues to increase as the glucose concentration rises to 10 mM or more. Under conditions of low blood glucose, the glucose concentration in a hepatocyte is low relative to the Km of hexokinase IV, and the glucose generated by gluconeogenesis leaves the cell before being trapped by phosphorylation. Second, hexokinase IV is not inhibited by glucose-6-phosphate, and it can therefore continue to operate when the accumulation of glucose 6-phosphate completely inhibits hexokinases I–III. Finally, hexokinase IV is subject to inhibition by the reversible binding of a regulatory protein specific to liver (Fig. 15– 13). The binding is much tighter in the presence of the allosteric effector fructose-6-phosphate. Glucose competes with fructose-6-phosphate for binding and causes dissociation of the regulatory protein from the hexokinase, relieving the inhibition. Immediately after a carbohydrate-rich meal, when blood glucose is high, glucose enters the hepatocyte via GLUT2 and activates hexokinase IV by this mechanism. During a fast, when blood glucose drops below 5 mM, fructose-6-phosphate triggers the inhibition of hexokinase IV by the regulatory protein, so the liver does not compete with other organs for the scarce glucose. The mechanism of inhibition by the regulatory protein is interesting: the protein anchors hexokinase IV inside the nucleus, where it is segregated from the other enzymes of glycolysis in the cytosol (Fig. 15–13). When the glucose concentration in the cytosol rises, it equilibrates with glucose in the nucleus by transport through the nuclear pores. Glucose causes dissociation of the regulatory protein, and hexokinase IV enters the cytosol and begins to phosphorylate glucose. 40 3.2.2 Transcriptional regulation of Hexokinase IV (Glucokinase) and Glucose-6-Phosphatase Hexokinase IV is also regulated at the level of protein synthesis. Circumstances that call for greater energy production (low [ATP], high [AMP], vigorous muscle contraction) or for greater glucose consumption (high blood glucose, for example) cause increased transcription of the hexokinase IV gene. Glucose-6-phosphatase, the gluconeogenic enzyme that bypasses the hexokinase step of glycolysis, is transcriptionally regulated by factors that call for increased production of glucose (low blood glucose, glucagon signaling). 3.2.3 Phosphofructokinase-1 and Fructose-1,6-bisphosphatase are Reciprocally Regulated Glucose 6-phosphate can flow either into glycolysis or through any of several other pathways, including glycogen synthesis and the pentose phosphate pathway. The metabolically irreversible reaction catalyzed by PFK-1 is the step that commits glucose to glycolysis. In addition to its substrate- binding sites, this complex enzyme has several regulatory sites at which allosteric activators or inhibitors bind. ATP is not only a substrate for PFK-1 but also an end product of the glycolytic pathway. When high cellular [ATP] signals that ATP is being produced faster than it is being consumed, ATP inhibits PFK- 1 by binding to an allosteric site and lowering the affinity of the enzyme for its substrate fructose-6- phosphate. ADP and AMP, which increase in concentration as consumption of ATP outpaces production, act allosterically to relieve this inhibition by ATP. These effects combine to produce higher enzyme activity when ADP or AMP accumulates and lower activity when ATP accumulates. Citrate (the ionized form of citric acid), a key intermediate in the aerobic oxidation of pyruvate, fatty acids, and amino acids, is also an allosteric regulator of PFK-1; high citrate concentration increases the inhibitory effect of ATP, further reducing the flow of glucose through glycolysis. In this case, citrate serves as an intracellular signal that the cell is meeting its current needs for energy-yielding metabolism by the oxidation of fats and proteins. The corresponding step in gluconeogenesis is the conversion of fructose-1,6-bisphosphate to fructose- 6-phosphate (Fig. 15–15). The enzyme that catalyzes this reaction, FBPase-1, is strongly inhibited 41 (allosterically) by AMP; when the cell’s supply of ATP is low (corresponding to high [AMP]), the ATP-requiring synthesis of glucose slows. Thus, these opposing steps in the glycolytic and gluconeogenic pathways—PFK-1 and FBPase-1—are regulated in a coordinated and reciprocal manner. In general, when sufficient concentrations of acetyl-CoA or citrate (the product of acetyl-CoA condensation with oxaloacetate) are present, or when a high proportion of the cell’s adenylate is in the form of ATP, gluconeogenesis is favored. When the level of AMP increases, it promotes glycolysis by stimulating PFK-1 (and also promotes glycogen degradation by activating glycogen phosphorylase). Summary of the regulators affecting PFK-1 activity. FIGURE 15–15: Regulation of fructose-1,6-bisphosphatase (FBPase-1) and phosphofructokinase-1 (PFK-1). 3.2.4 Fructose-2,6-Bisphosphate Is a Potent Allosteric Regulator of PFK-1 and FBPase-1 The special role of liver in maintaining a constant blood glucose level requires additional regulatory mechanisms to coordinate glucose production and consumption. When the blood glucose level decreases, the hormone glucagon signals the liver to produce and release more glucose and to stop consuming it for its own needs. One source of glucose is glycogen stored in the liver; another source is gluconeogenesis, using pyruvate, lactate, glycerol, or certain amino acids as starting material. When 42 blood glucose is high, insulin signals the liver to use glucose as a fuel and as a precursor for the synthesis and storage of glycogen and triacylglycerol. The rapid hormonal regulation of glycolysis and gluconeogenesis is mediated by fructose-2,6-bisphosphate, an allosteric effector for the enzymes PFK- 1 and FBPase-1: 3.2.5 Pyruvate Kinase is Allosterically Inhibited by ATP At least three isozymes of pyruvate kinase are found in vertebrates, differing in their tissue distribution and their response to modulators. High concentrations of ATP, acetyl-CoA, and long-chain fatty acids (signs of abundant energy supply) allosterically inhibit all isozymes of pyruvate kinase (Fig. 15–19). The liver isozyme (L form), but not the muscle isozyme (M form), is subject to further regulation by phosphorylation. When low blood glucose causes glucagon release, cAMP-dependent protein kinase phosphorylates the L isozyme of pyruvate kinase, inactivating it. This slows the use of glucose as a fuel in liver, sparing it for export to the brain and other organs. In muscle, the effect of increased [cAMP] is quite different. In response to epinephrine, cAMP activates glycogen breakdown and glycolysis and provides the fuel needed for the fight-or-flight response. 43 3.2.6 The Gluconeogenic Conversion of Pyruvate to Phosphoenol Pyruvate is Under Multiple Types of Regulation In the pathway leading from pyruvate to glucose, the first control point determines the fate of pyruvate in the mitochondrion: its conversion either to acetyl-CoA (by the pyruvate dehydrogenase complex) to fuel the citric acid cycle or to oxaloacetate (by pyruvate carboxylase) to start the process of gluconeogenesis. When fatty acids are readily available as fuels, their breakdown in liver mitochondria yields acetyl-CoA, a signal that further oxidation of glucose for fuel is not necessary. Acetyl-CoA is a positive allosteric modulator of pyruvate carboxylase and a negative modulator of pyruvate dehydrogenase, through stimulation of a protein kinase that inactivates the dehydrogenase. When the cell’s energy needs are being met, oxidative phosphorylation slows, NADH rises relative to NAD+ and inhibits the citric acid cycle, and acetyl-CoA accumulates. The increased concentration of acetyl-CoA inhibits the pyruvate dehydrogenase complex, slowing the formation of acetyl-CoA from pyruvate, and stimulates gluconeogenesis by activating pyruvate carboxylase, allowing conversion of excess pyruvate to oxaloacetate (and, eventually, glucose). Oxaloacetate formed in this way is converted to phosphoenolpyruvate (PEP) in the reaction catalyzed by PEP carboxykinase. In mammals, the regulation of this key enzyme occurs primarily at the level of its synthesis and 44 breakdown, in response to dietary and hormonal signals. Fasting or high glucagon levels act through cAMP to increase the rate of transcription and to stabilize the mRNA. Insulin, or high blood glucose, has the opposite effects. We discuss this transcriptional regulation in more detail below. Generally triggered by a signal from outside the cell (diet, hormones), these changes take place on a time scale of minutes to hours. 45 Numbering, pacing and Lesson 3 sequencing Title Gluconeogenesis, and Coordinated Regulation of Glycolysis and Gluconeogenesis Purpose To generally indulge deeper into carbohydrate metabolism; the pathway that lead to the generation of glucose from various precursors; Gluconeogenesis Brief summary of overall Please watch: Gluconeogenesis: task https://youtu.be/ydhr0QAyxYg Coordinated Regulation of Glycolysis and Gluconeogenesis: https://youtu.be/ardjd4h2Seo Spark Individual contribution Watch the video On the discussion forum, state the key steps in gluconeogenesis and its regulation. Interaction begins Post your answers on the discussion forum Look at your classmate’s responses. E-moderator interventions Providing feedback/ teaching points Summarising key points & closing the discussion Schedule and time This discussion should be done between 7th September, 2020 and 11th September, 2020 Next Glyoxylate cycle 46 4. The Glyoxylate Cycle 4.1 Introduction In plants, certain invertebrates, and some microorganisms (including E. coli and yeast) acetate can serve both as an energy-rich fuel and as a source of phosphoenolpyruvate for carbohydrate synthesis. In these organisms, enzymes of the glyoxylate cycle catalyze the net conversion of acetate to succinate or other four carbon intermediates of the citric acid cycle: In the glyoxylate cycle, acetyl-CoA condenses with oxaloacetate to form citrate, and citrate is converted to isocitrate, exactly as in the citric acid cycle. The next step, however, is not the breakdown of isocitrate by isocitrate dehydrogenase but the cleavage of isocitrate by isocitrate lyase, forming succinate and glyoxylate. The glyoxylate then condenses with a second molecule of acetyl-CoA to yield malate, in a reaction catalyzed by malate synthase. The malate is subsequently oxidized to oxaloacetate, which can condense with another molecule of acetyl-CoA to start another turn of the cycle. Each turn of the glyoxylate cycle consumes two molecules of acetyl-CoA and produces one molecule of succinate, which is then available for biosynthetic purposes. The succinate may be converted through fumarate and malate into oxaloacetate, which can then be converted to phosphoenolpyruvate by PEP carboxykinase, and thus to glucose by gluconeogenesis. Vertebrates do not have the enzymes specific to the glyoxylate cycle (isocitrate lyase and malate synthase) and therefore cannot bring about the net synthesis of glucose from lipids. In plants, the enzymes of the glyoxylate cycle are sequestered in membrane-bounded organelles called glyoxysomes, which are specialized peroxisomes. Those enzymes common to the citric acid and glyoxylate cycles have two isozymes, one specific to mitochondria, the other to glyoxysomes. Glyoxysomes are not present in all plant tissues at all times. They develop in lipid-rich seeds during germination, before the developing plant acquires the ability to make glucose by photosynthesis. In addition to glyoxylate cycle enzymes, glyoxysomes contain all the enzymes needed for the degradation of the fatty acids stored in seed oils. Acetyl-CoA formed from lipid breakdown is converted to succinate via the glyoxylate cycle, and the succinate is exported to mitochondria, where citric acid cycle enzymes transform it to malate. A cytosolic isozyme of malate dehydrogenase oxidizes malate to oxaloacetate, a precursor for gluconeogenesis. Germinating seeds can therefore convert the carbon of stored lipids into glucose. 47 Fig 1: Glyoxylate cycle. The citrate synthase, aconitase, and malate dehydrogenase of the glyoxylate cycle are isozymes of the citric acid cycle enzymes; isocitrate lyase and malate synthase are unique to the glyoxylate cycle. 4.2 The Citric Acid and Glyoxylate Cycles are Coordinately Regulated In germinating seeds, the enzymatic transformations of dicarboxylic and tricarboxylic acids occur in three intracellular compartments: mitochondria, glyoxysomes, and the cytosol. There is a continuous interchange of metabolites among these compartments. The carbon skeleton of oxaloacetate from the citric acid cycle (in the mitochondrion) is carried to the glyoxysome in the form of aspartate. Aspartate is converted to oxaloacetate, which condenses with acetyl-CoA derived from fatty acid breakdown. The citrate thus formed is converted to isocitrate by aconitase, and then split into glyoxylate and succinate by isocitrate lyase. The succinate returns to the mitochondrion, where it reenters the citric acid cycle and is transformed into malate, which enters the cytosol and is oxidized (by cytosolic malate dehydrogenase) to oxaloacetate. Oxaloacetate is converted via gluconeogenesis into hexoses and sucrose, which can be transported to the growing roots and shoot. Four distinct pathways participate in these conversions: a) fatty acid breakdown to acetyl-CoA (in glyoxysomes), b) the glyoxylate cycle (in glyoxysomes), 48  c) the citric acid cycle (in mitochondria) and d) gluconeogenesis (in the cytosol). Fig 2: Relationship between the glyoxylate and citric acid cycles. The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultaneously with, and mesh with, those of the citric acid cycle (in mitochondria), as intermediates pass between these compartments. The conversion of succinate to oxaloacetate is catalyzed by citric acid cycle enzymes. The sharing of common intermediates requires that these pathways be coordinately regulated. Isocitrate is a crucial intermediate, at the branch point between the glyoxylate and citric acid cycles. Isocitrate dehydrogenase is regulated by covalent modification: a specific protein kinase phosphorylates and thereby inactivates the dehydrogenase. This inactivation shunts isocitrate to the glyoxylate cycle, where it begins the synthetic route toward glucose. A phosphoprotein phosphatase removes the phosphoryl group from isocitrate dehydrogenase, reactivating the enzyme and sending more isocitrate through the energy-yielding citric acid cycle. The regulatory protein kinase and phosphoprotein phosphatase are separate enzymatic activities of a single polypeptide. 49 Fig 3: Coordinated regulation of glyoxylate and citric acid cycles. Regulation of isocitrate dehydrogenase activity determines the partitioning of isocitrate between the glyoxylate and citric acid cycles. When the enzyme is inactivated by phosphorylation (by a specific protein kinase), isocitrate is directed into biosynthetic reactions via the glyoxylate cycle. When the enzyme is activated by dephosphorylation (by a specific phosphatase), isocitrate enters the citric acid cycle and ATP is produced. Some bacteria, including E. coli, have the full complement of enzymes for the glyoxylate and citric acid cycles in the cytosol and can therefore grow on acetate as their sole source of carbon and energy. The phosphoprotein phosphatase that activates isocitrate dehydrogenase is stimulated by intermediates of the citric acid cycle and glycolysis and by indicators of reduced cellular energy supply. The same metabolites inhibit the protein kinase activity of the bifunctional polypeptide. Thus, the accumulation 50 of intermediates of the central energy-yielding pathways indicating energy depletion results in the activation of isocitrate dehydrogenase. When the concentration of these regulators falls, signaling a sufficient flux through the energy-yielding citric acid cycle, isocitrate dehydrogenase is inactivated by the protein kinase. The same intermediates of glycolysis and the citric acid cycle that activate isocitrate dehydrogenase are allosteric inhibitors of isocitrate lyase. When energy yielding metabolism is sufficiently fast to keep the concentrations of glycolytic and citric acid cycle intermediates low, isocitrate dehydrogenase is inactivated, the inhibition of isocitrate lyase is relieved, and isocitrate flows into the glyoxylate pathway, to be used in the biosynthesis of carbohydrates, amino acids, and other cellular components. 51 Numbering, Lesson 4 pacing and sequencing Title Glyoxylate cycle Purpose To generally indulge deeper into carbohydrate metabolism; the pathway that lead to the generation of glucose from lipids; Glyoxylate cycle Brief summary of Please watch: Glyoxylate cycle: https://youtu.be/pAG9ac-YL7M overall task Spark Individual Watch the video contribution On the discussion forum, state the key steps in glyoxylate. Interaction begins Post your answers on the discussion forum Look at your classmate’s responses. E-moderator Providing feedback/ teaching points interventions Summarising key points Closing the discussion Schedule and time This discussion should be done between 7th September, 2020 and 11th September, 2020 Next Hexose Monophosphate Pathway (HMP) 52 5. Lesson 5: Pentose Phosphate Pathway of Glucose Oxidation 5.0 Introduction In most animal tissues, the major catabolic fate of glucose 6-phosphate is glycolytic breakdown to pyruvate, much of which is then oxidized via the citric acid cycle which leads to ATP formation. Glucose 6-phosphate does have other catabolic fates, however, which lead to specialized products needed by the cell. Of particular importance in some tissues is the oxidation of glucose 6-phosphate to pentose phosphates by the pentose phosphate pathway also called the phosphogluconate pathway or the hexose monophosphate pathway. In this oxidative pathway, NADP+ is the electron acceptor, yielding NADPH. Rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa, and those of tumors, use the pentose ribose 5-phosphate to make RNA, DNA, and such coenzymes as ATP, NADH, FADH2, and coenzyme A. In other tissues, the essential product of the pentose phosphate pathway is not the pentoses but the electron donor NADPH, needed for reductive biosynthesis or to counter the damaging effects of oxygen radicals. Tissues that carry out extensive fatty acid synthesis (liver, adipose, lactating mammary gland) or very active synthesis of cholesterol and steroid hormones (liver, adrenal glands, gonads) require the NADPH provided by this pathway. Erythrocytes and the cells of the lens and cornea are directly exposed to oxygen and thus to the damaging free radicals generated by oxygen. By maintaining a reducing atmosphere (a high ratio of NADPH to NADP+ and a high ratio of reduced to oxidized glutathione), such cells can prevent or undo oxidative damage to proteins, lipids, and other sensitive molecules. In erythrocytes, the NADPH produced by the pentose phosphate pathway is so important in preventing oxidative damage that a genetic defect in glucose 6-phosphate dehydrogenase, the first enzyme of the pathway, can have serious medical consequences. 53  Fig 1: General scheme of the pentose phosphate pathway 5.1 The Oxidative Phase Produces Pentose Phosphates and NADPH The first reaction of the pentose phosphate pathway is the oxidation of glucose 6-phosphate by glucose 6-phosphate dehydrogenase (G6PD) to form 6-phosphoglucono-δ-lactone, an intramolecular ester. NADP+ is the electron acceptor, and the overall equilibrium lies far in the direction of NADPH formation. The lactone is hydrolyzed to the free acid 6-phosphogluconate by a specific lactonase, then 6-phosphogluconate undergoes oxidation and decarboxylation by 6-phosphogluconate dehydrogenase to form the ketopentose ribulose 5-phosphate; the reaction generates a second molecule of NADPH. (This ribulose 5-phosphate is important in the regulation of glycolysis and gluconeogenesis. Phosphopentose isomerase converts ribulose 5-phosphate to its aldose isomer, ribose 5-phosphate. In some tissues, the pentose phosphate pathway ends at this point, and its overall equation is: The net result is the production of NADPH, a reductant for biosynthetic reactions, and ribose 5- phosphate, a precursor for nucleotide synthesis. 54 Fig 2: Oxidative reactions of the pentose phosphate pathway; the end products are ribose 5- phosphate, CO2, and NADPH. 55 5.2 The Nonoxidative Phase Recycles Pentose Phosphates to Glucose 6-Phosphate In tissues that require primarily NADPH, the pentose phosphates produced in the oxidative phase of the pathway are recycled into glucose 6-phosphate. In this nonoxidative phase, ribulose 5-phosphate is first epimerized to xylulose 5-phosphate: Then, in a series of rearrangements of the carbon skeletons, six five-carbon sugar phosphates are converted to five six-carbon sugar phosphates, completing the cycle and allowing continued oxidation of glucose 6-phosphate with production of NADPH. Continued recycling leads ultimately to the conversion of glucose 6-phosphate to six CO2. Two enzymes unique to the pentose phosphate pathway act in these interconversions of sugars: transketolase and transaldolase. Fig 3: Nonoxidative reactions of the pentose phosphate pathway; (a) these reactions convert pentose phosphates to hexose phosphates, allowing the oxidative reactions to continue. 56 Transketolase and transaldolase are specific to this pathway; the other enzymes also serve in the glycolytic or gluconeogenic pathways. (b) A schematic diagram showing the pathway from six pentoses (5C) to five hexoses (6C). Note that this involves two sets of the interconversions shown in (a). Every reaction shown here is reversible; unidirectional arrows are used only to make clear the direction of the reactions during continuous oxidation of glucose 6-phosphate. In the light independent reactions of photosynthesis, the direction of these reactions is reversed. Transketolase catalyzes the transfer of a two-carbon fragment from a ketose donor to an aldose acceptor. In its first appearance in the pentose phosphate pathway, transketolase transfers C-1 and C- 2 of xylulose 5-phosphate to ribose 5-phosphate, forming the seven-carbon product sedoheptulose 7- phosphate. The remaining three-carbon fragment from xylulose is glyceraldehyde 3-phosphate. Fig 4: The first reaction catalyzed by transketolase. (a) The general reaction catalyzed by transketolase is the transfer of a two-carbon group, carried temporarily on enzyme-bound TPP, from a ketose donor to an aldose acceptor. (b) Conversion of two pentose phosphates to a triose phosphate and a seven-carbon sugar phosphate, sedoheptulose 7-phosphate. 57 Next, transaldolase catalyzes a reaction similar to the aldolase reaction of glycolysis: a three-carbon fragment is removed from sedoheptulose 7-phosphate and condensed with glyceraldehyde 3- phosphate, forming fructose 6-phosphate and the tetrose erythrose 4-phosphate. Now transketolase acts again, forming fructose 6-phosphate and glyceraldehyde 3-phosphate from erythrose 4-phosphate and xylulose 5-phosphate. Two molecules of glyceraldehyde 3-phosphate formed by two iterations of these reactions can be converted to a molecule of fructose 1,6-bisphosphate as in gluconeogenesis, and finally FBPase-1 and phosphohexose isomerase convert fructose 1,6-bisphosphate to glucose 6-phosphate. Overall, six pentose phosphates have been converted to five hexose phosphates. Transketolase requires the cofactor thiamine pyrophosphate (TPP), which stabilizes a two-carbon carbanion in this reaction, just as it does in the pyruvate decarboxylase reaction. Transaldolase uses a Lys side chain to form a Schiff base with the carbonyl group of its substrate, a ketose, thereby stabilizing a carbanion that is central to the reaction mechanism. 58  HEALTH BYTE: Wernicke-Korsakoff syndrome is exacerbated by a defect in transketolase. Wernicke-Korsakoff syndrome is a disorder caused by a severe deficiency of thiamine, a component of TPP. The syndrome is more common among people with alcoholism than in the general population, because chronic, heavy alcohol consumption interferes with the intestinal absorption of thiamine. The syndrome can be exacerbated by a mutation in the gene for transketolase that results in an enzyme with a lowered affinity for TPP—an affinity one-tenth that of the normal enzyme. This defect makes individuals much more sensitive to a thiamine deficiency: even a moderate thiamine deficiency (tolerable in individuals with an unmutated transketolase) can drop the level of TPP below that needed to saturate the enzyme. The result is a slowing down of the whole pentose phosphate pathway. In people with Wernicke-Korsakoff syndrome this results in a worsening of symptoms, which can include severe memory loss, mental confusion, and partial paralysis. 59 Numbering, Lesson 5 pacing and sequencing Title Hexose Monophosphate Pathway (HMP) Purpose To generally indulge deeper into carbohydrate metabolism; Hexose Monophosphate Pathway (HMP) Brief summary of Please watch: Hexose Monophosphate Pathway (HMP): overall task https://youtu.be/bM9T6MMp7bA Spark Individual Watch the video contribution On the discussion forum, state the key steps in PPP. Interaction begins Post your answers on the discussion forum Look at your classmate’s responses. E-moderator Providing feedback/ teaching points interventions Summarising key points Closing the discussion Schedule and time This discussion should be done between 7th September, 2020 and 11th September, 2020 Next Glycogen metabolism and its regulation 60 6. Lesson 6: The Metabolism of Glycogen in Animals 6.0 Introduction In organisms from bacteria to plants to vertebrates, excess glucose is converted to polymeric forms for storage; glycogen in vertebrates and many microorganisms, starch in plants. In vertebrates, glycogen is found primarily in the liver and skeletal muscle; it may represent up to 10% of the weight of liver and 1% to 2% of the weight of muscle. The glycogen in muscle is there to provide a quick source of energy for either aerobic or anaerobic metabolism. Muscle glycogen can be exhausted in less than an hour during vigorous activity. Liver glycogen serves as a reservoir of glucose for other tissues when dietary glucose is not available (between meals or during a fast); this is especially important for the neurons of the brain, which cannot use fatty acids as fuel. Liver glycogen can be depleted in 12 to 24 hours. In humans, the total amount of energy stored as glycogen is far less than the amount stored as fat (triacylglycerol), but fats cannot be converted to glucose in mammals and cannot be catabolized anaerobically. Glycogen granules are complex aggregates of glycogen and the enzymes that synthesize it and degrade it, as well as the machinery for regulating these enzymes. The general mechanisms for storing and mobilizing glycogen are the same in muscle and liver, but the enzymes differ in subtle yet important ways that reflect the different roles of glycogen in the two tissues. Glycogen is also obtained in the diet and broken down in the gut, and this involves a separate set of hydrolytic enzymes that convert glycogen to free glucose. 6.1 Glycogen Breakdown is Catalyzed by Glycogen Phosphorylase In skeletal muscle and liver, the glucose units of the outer branches of glycogen enter the glycolytic pathway through the action of three enzymes: glycogen phosphorylase, glycogen debranching enzyme, and phosphoglucomutase. Glycogen phosphorylase catalyzes the reaction in which an (α1→4) glycosidic linkage between two glucose residues at a nonreducing end of glycogen undergoes attack by inorganic phosphate (Pi), removing the terminal glucose residue as α-D-glucose 1-phosphate. 61 This phosphorolysis reaction is different from the hydrolysis of glycosidic bonds by amylase during intestinal degradation of dietary glycogen and starch. In phosphorolysis, some of the energy of the glycosidic bond is preserved in the formation of the phosphate ester, glucose 1-phosphate. Pyridoxal phosphate is an essential cofactor in the glycogen phosphorylase reaction; its phosphate group acts as a general acid catalyst, promoting attack by Pi on the glycosidic bond. Glycogen phosphorylase acts repetitively on the nonreducing ends of glycogen branches until it reaches a point four glucose residues away from an (α1→6) branch point, where its action stops. Further degradation by glycogen phosphorylase can occur only after the debranching enzyme, formally known as oligo (α1→6) to (α1→4) glucan-transferase, catalyzes two successive reactions that transfer branches. Once these branches are transferred and the glucosyl residue at C-6 is hydrolyzed, glycogen phosphorylase activity can continue. 62 Figure 1: Glycogen breakdown near an (a1→6) branch point. Following sequential removal of terminal glucose residues by glycogen phosphorylase, glucose residues near a branch are removed in a two-step process that requires a bifunctional debranching enzyme. First, the transferase activity of the enzyme shifts a block of three glucose residues from the branch to a nearby nonreducing end, to which they are reattached in (α1→4) linkage. The single glucose residue remaining at the branch point, in (α1→6) linkage, is then released as free glucose by the debranching enzyme’s (α1→6) glucosidase activity. The glucose residues are shown in shorthand form, which omits the ⎯H, ⎯OH, and ⎯CH2OH groups from the pyranose rings. 6.2 Glucose 1-Phosphate Can Enter Glycolysis or, in Liver, Replenish Blood Glucose Glucose 1-phosphate, the end product of the glycogen phosphorylase reaction, is converted to glucose 6-phosphate by phosphoglucomutase, which catalyzes the reversible reaction below: 63 Initially phosphorylated at a Ser residue, the enzyme donates a phosphoryl group to C-6 of the substrate, then accepts a phosphoryl group from C-1. The glucose 6-phosphate formed from glycogen in skeletal muscle can enter glycolysis and serve as an energy source to support muscle contraction. In liver, glycogen breakdown serves a different purpose: to release glucose into the blood when the blood glucose level drops, as it does between meals. This requires the enzyme glucose 6-phosphatase, present in liver and kidney but not in other tissues. The enzyme is an integral membrane protein of the endoplasmic reticulum, predicted to contain nine transmembrane helices, with its active site on the lumenal side of the ER. Glucose 6-phosphate formed in the cytosol is transported into the ER lumen by a specific transporter (T1) and hydrolyzed at the lumenal surface by the glucose 6-phosphatase. The resulting Pi and glucose are thought to be carried back into the cytosol by two different transporters (T2 and T3), and the glucose leaves the hepatocyte via the plasma membrane transporter, GLUT2. By having the active site of glucose 6-phosphatase inside the ER lumen, the cell separates this reaction from the process of glycolysis, which takes place in the cytosol and would be aborted by the action of glucose 6-phosphatase. Genetic defects in either glucose 6-phosphatase or T1 lead to serious derangement of glycogen metabolism, resulting in type Ia glycogen storage disease. Because muscle and adipose tissue lack glucose 6- phosphatase, they cannot convert the glucose 6- phosphate formed by glycogen breakdown to glucose, and these tissues therefore do not contribute glucose to the blood. 64 6.3 UDP-Glucose Involvement with Glycogen Synthesis Many of the reactions in which hexoses are transformed or polymerized involve sugar nucleotides, compounds in which the anomeric carbon of a sugar is activated by attachment to a nucleotide through a phosphate ester linkage. Sugar nucleotides are the substrates for polymerization of monosaccharides into disaccharides, glycogen, starch, cellulose, and more complex extracellular polysaccharides. They are also key intermediates in the production of the aminohexoses and deoxyhexoses found in some of these polysaccharides, and in the synthesis of vitamin C (L-ascorbic acid). 65 The suitability of sugar nucleotides for biosynthetic reactions stems from several properties: 1. Their formation is metabolically irreversible, contributing to the irreversibility of the synthetic pathways in which they are intermediates. The condensation of a nucleoside triphosphate with a hexose 1-phosphate to form a sugar nucleotide has a small positive free-energy change, but the reaction releases PPi, which is rapidly hydrolyzed by inorganic pyrophosphatase, in a reaction that is strongly exergonic. This keeps the cellular concentration of PPi low, ensuring that the actual free-energy change in the cell is favorable. In effect, rapid removal of the product, driven by the large, negative free-energy chang