Carbohydrates PDF

CARBOHYDRATES PRESENTED BY DR. RANIA ELLETHY I. CARBOHYDRATES ‫اﻟﻤﺤﺎﺿﺮة اﻻوﻟﻰ‬ INTRODUCTION TO CARBOHYDRATES *Carbohydrates are the most abundant organic molecules in nature. *The empiric formula for many of the simpler carbohydrates is (CH2O)n, hence the name “hydrate of carbon.” *functions o...

CARBOHYDRATES PRESENTED BY DR. RANIA ELLETHY I. CARBOHYDRATES ‫اﻟﻤﺤﺎﺿﺮة اﻻوﻟﻰ‬ INTRODUCTION TO CARBOHYDRATES *Carbohydrates are the most abundant organic molecules in nature. *The empiric formula for many of the simpler carbohydrates is (CH2O)n, hence the name “hydrate of carbon.” *functions of carbohydrates: 1) It is the main source of energy where about 60% of energy which needed by the cell is obtained from carbohydrates metabolism. 2) serving as cell membrane components that mediate some forms of intercellular communication. 3) Carbohydrates also serve as a structural component of many organisms, including the cell walls of bacteria, the exoskeleton of many insects, and the fibrous cellulose of plants. DEFINITION:  Carbohydrates are poly hydroxy ketenes or poly hydroxy aldehydes, contain either aldehyde or Ketone group as a functional group. *Carbohydrates with an aldehyde as their most oxidized functional group are called aldoses, whereas those with a keto as their most oxidized functional group are called ketoses (Figure 7.2). CLASSIFICATION OF CARBOHYDRATES:- Monosaccharides (simple sugars) contain two monosaccharide units. Important disaccharides include lactose Disaccharides (galactose + glucose), sucrose (glucose + fructose), and maltose (glucose + glucose). contain from three to about ten monosaccharide Oligosaccharides units. contain more than ten monosaccharide units, and can be hundreds of sugar units in length. Important polysaccharides include branched glycogen (from animal sources) and starch Polysaccharides (plant sources) and unbranched cellulose (plant sources); each is a polymer of glucose. The bonds that link sugars are called glycosidic bonds. These are formed by enzymes known as glycosyltransferases.  MONOSACCHARIDES:  Physical properties: 1) Colorless solution. 2) Soluble in water. 3) Crystalline shape. 4) Most of it has sweet test.  classification of monosaccharides according to the number of carbons they contain *ISOMERS AND EPIMERS  Isomers, Compounds that have the same chemical formula but have different structures. For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, C6H12O6.  Epimers, Carbohydrate isomers that differ in configuration around only one specific carbon atom are defined as epimers of each other. For example, glucose and galactose are C-4 epimers—their structures differ only in the position of the –OH group at carbon 4. Glucose and mannose are C-2 epimers.  Figure 3: C-2 and C-4 epimers and an isomer of glucose. *ENANTIOMERS  special type of isomerism is found in the pairs of structures that are mirror images of each other. These mirror images are called enantiomers, and the two members of the pair are designated as a D- and an L- sugar (Figure 4).  *The vast majority of the sugars in humans are D- sugars.  In the D isomeric form, the –OH group on the asymmetric carbon (a carbon linked to four different atoms or groups) farthest from the carbonyl carbon is on the right, whereas in the L-isomer it is on the left. *Enzymes known as racemases are able to interconvert D- and L-isomers.  Figure 4: Enantiomers (mirror images) of glucose. *CYCLIZATION OF MONOSACCHARIDES:  Less than 1% of each of the monosaccharides with five or more carbons exists in the open-chain (acyclic) form.  Rather, they are predominantly found in a ring (cyclic) form, in which the aldehyde (or keto) group has reacted with an alcohol group on the same sugar, making the carbonyl carbon (carbon 1 for an aldose or carbon 2 for a ketose) asymmetric.  [Note: Pyranose refers to a six-membered ring consisting of five carbons and one oxygen, for example, glucopyranose (Figure 5), whereas furanose denotes a five-membered ring with four carbons and one oxygen.]  1.ANOMERIC CARBON:  Cyclization creates an anomeric carbon (the former carbonyl carbon), generating the α and β configurations of the sugar, for example, α-D glucopyranose and β-D-glucopryanose (see Figure 6).  These two sugars are both glucose but are anomers of each other.  [Note: In the α configuration, the OH on the anomeric C projects to the same side as the ring in a modified Fischer projection formula (Figure 6A), and is trans to the CH2OH group in a Haworth projection formula (Figure 6B).Because the α and β forms are not mirror images, they are referred to as diastereomers.  Figure 6: A The interconversion (mutarotation) of the α and β anomeric forms of glucose shown as modified Fischer projection formulas. B. The interconversion shown as Haworth projection formulas. Carbon 1 is the anomeric carbon. [Note: Glucose is a reducing sugar.] 2. REDUCING SUGARS:  If the hydroxyl group on the anomeric carbon of a cyclized sugar is not linked to another compound by a glycosidic bond, the ring can open. The sugar can act as a reducing agent, and is termed a reducing sugar.  Such sugars can react with chromogenic agents (for example, Benedict’s reagent or Fehling’s solution) causing the reagent to be reduced and colored, with the aldehyde group of the acyclic sugar becoming oxidized.  [Note: Only the state of the oxygen in the aldehyde group determines if the sugar is reducing or non- reducing.] ‫اﻟﻤﺤﺎﺿﺮة اﻟﺜﺎﻧﯿﺔ‬ DIGESTION OF DIETARY CARBOHYDRATES  The principal sites of dietary carbohydrate digestion are the mouth and intestinal lumen.  This digestion is rapid and is catalyzed by enzymes known as glycoside hydrolases (glycosidases) that hydrolyze glycosidic bonds.  Because there is little monosaccharide present in diets of mixed animal and plant origin, the enzymes are primarily endoglycosidases that hydrolyze polysaccharides and oligosaccharides, and disaccharidases that hydrolyse tri- and disaccharides into their reducing sugar components (Figure 7).  The final products of carbohydrate digestion are the monosaccharides, glucose, galactose and fructose, which are absorbed by cells of the small intestine.  Figure 7: Hydrolysis of a glycosidic bond.  A. DIGESTION OF CARBOHYDRATES BEGINS IN THE MOUTH:  The major dietary polysaccharides are of plant (starch, composed of amylose and amylopectin) and animal (glycogen) origin.  During mastication, salivary α-amylase acts briefly on dietary starch and glycogen, hydrolyzing random α(1→4) bonds.  [Note: There are both α(1→4)- and β(1→4)-endoglucosidases in nature, but humans do not produce the latter. Therefore, we are unable to digest cellulose— a carbohydrate of plant origin containing β(1→4) glycosidic bonds between glucose residues.]  [Note: Disaccharides are also present as they, too, are resistant to amylase.]  Carbohydrate digestion halts temporarily in the stomach, because the high acidity inactivates salivary α-amylase. FIGURE 8: DEGRADATION OF DIETARY GLYCOGEN BY SALIVARY OR PANCREATIC Α- AMYLASE. B. FURTHER DIGESTION OF CARBOHYDRATES BY PANCREATIC ENZYMES OCCURS IN THE SMALL INTESTINE: *When the acidic stomach contents reach the small intestine, they are neutralized by bicarbonate secreted by the pancreas, and pancreatic α-amylase continues the process of starch digestion. C. FINAL CARBOHYDRATE DIGESTION BY ENZYMES SYNTHESIZED BY THE INTESTINAL MUCOSAL CELLS:  The final digestive processes occur primarily at the mucosal lining of the upper jejunum, and include the action of several disaccharidases (Figure 9).  For example, isomaltase cleaves the α(1→6) bond in isomaltose and maltase cleaves maltose and maltotriose, each producing glucose, sucrase cleaves sucrose producing glucose and fructose, and lactase (β-galactosidase) cleaves lactose producing galactose and glucose.  These enzymes are secreted through, and remain associated with, the luminal side of the brush border membranes of the intestinal mucosal cells. FIGURE 9: DIGESTION OF CARBOHYDRATE. [NOTE: INDIGESTIBLE CELLULOSE ENTERS THE COLON AND IS EXCRETED.] D. ABSORPTION OF MONOSACCHARIDES BY INTESTINAL MUCOSAL CELLS:  The duodenum and upper jejunum absorb the bulk of the dietary sugars. However, different sugars have different mechanisms of absorption.  For example, galactose and glucose are transported into the mucosal cells by an active, energy-requiring process that requires a concurrent uptake of sodium ions; the transport protein is the sodium-dependent glucose cotransporter 1 (SGLT- 1).  Fructose uptake requires a sodium-independent monosaccharide transporter (GLUT-5) for its absorption.  All three monosaccharides are transported from the intestinal mucosal cell into the portal circulation by yet another transporter, GLUT-2. E. ABNORMAL DEGRADATION OF DISACCHARIDES:  The overall process of carbohydrate digestion and absorption is so efficient in healthy individuals that ordinarily all digestible dietary carbohydrate is absorbed by the time the ingested material reaches the lower jejunum.  However, because it is mono saccharides that are absorbed, any defect in a specific disaccharidase activity of the intestinal mucosa causes the passage of undigested carbohydrate into the large intestine.  As a consequence of the presence of this osmotically active material, water is drawn from the mucosa into the large intestine, causing osmotic diarrhea. This is reinforced by the bacterial fermentation of the remaining carbohydrate to two- and three-carbon compounds (which are also osmotically active) plus large volumes of CO2 and H2 gas, causing abdominal cramps, diarrhea, and flatulence.  1. Digestive enzyme deficiencies:  Genetic deficiencies of the individual disaccharidases result in disaccharide intolerance.  Alterations in disaccharide degradation can also be caused by a variety of intestinal diseases, malnutrition, or drugs that injure the mucosa of the small intestine.  2. Lactose intolerance:  More than three quarters of the world’s adults are lactose intolerant (Figure 10). This is particularly manifested in certain populations.  For example, up to 90% of adults of African or Asian descent are lactase-deficient and, therefore, are less able to metabolize lactose than individuals of Northern European origin.  The age-dependent loss of lactase activity represents a reduction in the amount of enzyme rather than a modified inactive enzyme.  It is thought to be caused by small variations in the DNA sequence of a region on chromosome 2 that controls expression of the gene for lactase, also on chromosome 2.  Treatment for this disorder is to reduce consumption of milk while eating yogurts and cheeses, as well as green vegetables such as broccoli, to ensure adequate calcium intake; to use lactase-treated products; or to take lactase in pill form prior to eating.  [Note: Because the loss of lactase is the norm for most of the world’s adults, use of the term “adult hypolactasia” for lactose intolerance is becoming more common.] Figure 10: Abnormal lactose metabolism. ‫اﻟﻤﺤﺎﺿﺮة اﻟﺜﺎﻟﺜﺔ‬  GLYCOLYSIS I. INTRODUCTION TO METABOLISM:  metabolism, which is the sum of all the chemical metabolism, changes occurring in a cell, a tissue, or the body.  Most pathways can be classified as either catabolic (degradative) or anabolic (synthetic).  Catabolic reactions break down complex molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules, for example, CO2, NH3 (ammonia), and water.  Anabolic pathways form complex end products from simple precursors, for example, the synthesis of the polysaccharide, glycogen, from glucose. Figure 11: Important reactions of intermediary metabolism. Blue text = intermediates of carbohydrate metabolism; brown text = intermediates of lipid metabolism; green text = intermediates of protein metabolism. A. Catabolic pathways:-  Catabolic reactions serve to capture chemical energy in the form of adenosine triphosphate (ATP) from the degradation of energy-rich fuel molecules.  Catabolism also allows molecules in the diet (or nutrient molecules stored in cells) to be converted into building blocks needed for the synthesis of complex molecules.  Energy generation by degradation of complex molecules occurs in three stages as shown in Figure 12.  [Note: Catabolic pathways are typically oxidative, and require coenzymes such as NAD+.] Figure 12: Three stages of catabolism. *THREE STAGES OF CATABOLISM: 1.Hydrolysis of complex molecules: In the first stage, complex molecules are broken down into their component building blocks. For example, proteins are degraded to amino acids, polysaccharides to monosaccharides, and fats (triacylglycerols) to free fatty acids and glycerol. 2.Conversion of building blocks to simple intermediates: In the second stage, these diverse building blocks are further degraded to acetyl coenzyme A (CoA) and a few other, simple molecules. Some energy is captured as ATP, but the amount is small compared with the energy produced during the third stage of catabolism. 3.Oxidation of acetyl CoA: The tricarboxylic acid (TCA) cycle is the final common pathway in the oxidation of fuel molecules that produce acetyl CoA. Oxidation of acetyl CoA generates large amounts of ATP via oxidative phosphorylation as electrons flow from NADH and FADH2 to oxygen. B. Anabolic pathways:  Anabolic reactions combine small molecules, such as amino acids, to form complex molecules, such as proteins (Figure 13).  Anabolic reactions require energy (are endergonic), which is generally provided by the breakdown of ATP to adenosine diphosphate (ADP) and inorganic phosphate (Pi).  Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH.  Note that  catabolism is a convergent process—that is, a wide variety of molecules are transformed into a few common end products. By contrast,  anabolism is a divergent process in which a few biosynthetic precursors form a wide variety of polymeric or complex products. Figure 13: Comparison of catabolic and anabolic pathways. II. REGULATION OF METABOLISM:  The pathways of metabolism must be coordinated so that the production of energy or the synthesis of end products meets the needs of the cell.  Furthermore, individual cells do not function in isolation but, rather, are part of a community of interacting tissues. Thus, a communication system has evolved to coordinate the functions of the body.  Regulatory signals that inform an individual cell of the metabolic state of the body as a whole include hormones, neurotransmitters, and the availability of nutrients. These, in turn, influence signals generated within the cell (Figure 14). Figure 14: Some commonly used mechanisms for transmission of regulatory signals between cells. A. Signals from within the cell (intracellular):  The rate of a metabolic pathway can respond to regulatory signals that arise from within the cell.  For example, the rate of a pathway may be influenced by the availability of substrates, product inhibition, or alterations in the levels of allosteric activators or inhibitors.  These intracellular signals typically elicit rapid responses, and are important for the moment-to- moment regulation of metabolism. B. Communication between cells (intercellular):  The ability to respond to extracellular signals is essential for the survival and development of all organisms.  Signaling between cells provides for long-range integration of metabolism, and usually results in a response that is slower than is seen with signals that originate within the cell.  Communication between cells can be mediated, for example, by surface-to-surface contact and, in some tissues, by formation of gap junctions, allowing direct communication between the cytoplasms of adjacent cells. However, for energy metabolism, the most important route of communication is chemical signaling between cells by bloodborne hormones or by neurotransmitters. III. OVERVIEW OF GLYCOLYSIS:  The glycolytic pathway is employed by all tissues for the breakdown of glucose to provide energy (in the form of ATP) and intermediates for other metabolic pathways.  Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars—whether arising from the diet or from catabolic reactions in the body—can ultimately be converted to glucose (Figure 15A).  Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate supply of oxygen.  This series of ten reactions is called aerobic glycolysis because oxygen is required to reoxidize the NADH formed during the oxidation of glyceraldehyde 3- phosphate (Figure 15B).  Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the TCA (or citric acid) cycle.  Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD+ (Figure 15C). This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of oxygen.  Anaerobic glycolysis allows the production of ATP in tissues that lack mitochondria (for example, red blood cells) or in cells deprived of sufficient oxygen. Figure 15: A. Glycolysis pathways. B. Reactions of aerobic glycolysis. C. Reactions of anaerobic glycolysis. IV. TRANSPORT OF GLUCOSE INTO CELLS:  Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms: A- Na+-independent facilitated diffusion transport system. B- or Na+-monosaccharide cotransporter system. A. Na+-independent facilitated diffusion transport:-  This system is mediated by a family of 14 glucose transporters in cell membranes. They are designated GLUT-1 to GLUT-14 (glucose transporter isoforms 1– 14).  These transporters exist in the membrane in two conformational states (Figure 16).  Extra cellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane. Figure 16: Schematic representation of the facilitated transport of glucose through a cell membrane. [Note: GLUT proteins contain 12 tran-smembrane helices.] B. Na+-monosaccharide cotransporter system:-  This is an energy-requiring process that transports glucose “against” a concentration gradient—that is, from low glucose concentrations outside the cell to higher concentrations within the cell.  This system is a carrier-mediated process in which the movement of glucose is coupled to the concentration gradient of Na+, which is transported into the cell at the same time.  The carrier is a sodium-dependent– glucose transporter or SGLT. This type of transport occurs in the epithelial cells of the intestine, renal tubules, and choroid plexus. ‫اﻟﻤﺤﺎﺿﺮة اﻟﺮاﺑﻌﺔ‬ V. REACTIONS OF GLYCOLYSIS: Figure: Glycolysis metabolic pathway.  The conversion of glucose to pyruvate occurs in two stages (Figure 18):  The first five reactions of glycolysis correspond to an energy investment phase in which the phosphorylated forms of intermediates are synthesized at the expense of ATP.  The subsequent reactions of glycolysis constitute an energy generation phase in which a net of two molecules of ATP are formed by substrate-level phosphorylation per glucose molecule metabolized. Figure 18: Two phases of aerobic glycolysis. A. Phosphorylation of glucose  Phosphorylated sugar molecules do not readily penetrate cell membranes, because there are no specific transmembrane carriers for these compounds, and because they are too polar to diffuse through the lipid core of membranes.  The irreversible phosphorylation of glucose (Figure 19), therefore, effectively traps the sugar as cytosolic glucose 6- phosphate, thus committing it to further metabolism in the cell.  Mammals have several isozymes of the enzyme hexokinase that catalyze the phosphorylation of glucose to glucose 6- Figure 19: Energy investment phosphate. phase: phosphorylation of glucose. 1. Hexokinase: In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three regulatory enzymes of glycolysis.  Hexokinase has broad substrate specificity and is able to phosphorylate several hexoses in addition to glucose.  Hexokinase is inhibited by the reaction product, glucose 6- phosphate, which accumulates when further metabolism of this hexose phosphate is reduced.  Hexokinase has a low Km for glucose. This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low.  [Note: Hexokinase also serves as a glucose sensor in neurons of the hypothalamus, playing a key role in the adrenergic response to hypoglycemia]  2. Glucokinase:  In liver parenchymal cells and β cells of the pancreas, glucokinase (also called hexokinase D, or type IV) is the predominant enzyme responsible for the phosphorylation of glucose.  In β cells, glucokinase functions as the glucose sensor, determining the threshold for insulin secretion. In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia.  glucokinase functions only when the intracellular concentration of glucose in the hepatocyte is elevated, such as during the brief period following consumption of a carbohydrate- rich meal, when high levels of glucose are delivered to the liver via the portal vein.  Glucokinase has a high Vmax, allowing the liver to effectively remove the flood of glucose delivered by the portal blood. This prevents large amounts of glucose from entering the systemic circulation following a carbohydrate rich meal, and thus minimizes hyperglycemia during the absorptive period. Figure 20: Effect of glucose concentration on the rate of phosphorylation catalyzed by hexokinase and glucokinase. B. Isomerization of glucose 6- phosphate:  The isomerization of glucose 6- phosphate to fructose 6- phosphate is catalyzed by phospho glucose isomerase (Figure 21).  The reaction is readily reversible and is not a rate-limiting or regulated step. Figure 21: Aldose-ketose isomerization of glucose 6-phosphate to fructose 6-phosphate. C. Phosphorylation of fructose 6-phosphate  The irreversible phosphorylation reaction catalyzed by phospho - fructokinase-1 (PFK-1) is the most important control point and the rate- limiting and committed step of glycolysis (Figure 22).  PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6- phosphate, and by regulatory substances described below. Figure 22: Energy investment phase (continued): Conversion of fructose 6- phosphate to triose phosphates. 1. Regulation by energy levels within the cell: * PFK-1 is inhibited allosterically by elevated levels of ATP, which act as an “energy rich” signal indicating an abundance of high-energy compounds.  Elevated levels of citrate, an intermediate in the TCA cycle, also inhibit PFK-1.  Conversely, PFK-1 is activated allosterically by high concentrations of AMP. 2. Regulation by fructose 2,6-bisphosphate: Fructose 2,6-bisphosphate is the most potent activator of PFK-1, and is able to activate the enzyme even when ATP levels are high.  Fructose 2,6-bisphosphate is formed by phosphofructokinase-2 (PFK-2), an enzyme different than PFK-1.  PFK-2 is a bifunctional protein that has both the kinase activity that produces fructose 2,6-bisphosphate and a phosphatase activity that dephosphorylates fructose 2,6-bisphosphate back to fructose 6-phosphate.  In liver, the kinase domain is active if dephosphorylated and is inactive if phosphorylated. a. During the well-fed state:  Decreased levels of glucagon and elevated levels of insulin, such as occur following a carbohydrate- rich meal, cause an increase in fructose 2,6-bisphosphate and, thus, in the rate of glycolysis in the liver.  Fructose 2,6-bisphosphate, therefore, acts as an intracellular signal, indicating that glucose is abundant. b. During starvation: Elevated levels of glucagon and low levels of insulin, such as occur during fasting , decrease the intracellular concentration of hepatic fructose 2,6-bisphosphate.  This results in a decrease in the overall rate of glycolysis and an increase in gluconeogenesis. D. Cleavage of fructose 1,6-bisphosphate  Aldolase cleaves fructose 1,6-bisphosphate to dihydroxy acetone phosphate and glyceraldehyde 3-phosphate (see Figure 22).  The reaction is reversible and not regulated. E. Isomerization of dihydroxyacetone phosphate  Triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 22).  Dihydroxy-acetone phosphate must be isomerized to glyceraldehyde 3-phosphate for further metabolism by the glycolytic pathway.  This isomerization results in the net production of two molecules of glyceraldehyde 3-phosphate from the cleavage products of fructose 1,6- bisphosphate. F. Oxidation of glyceraldehyde 3-phosphate  The conversion of glyceraldehyde 3-phosphate to 1,3- bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis (Figure 23).  [Note: Because there is only a limited amount of NAD+ in the cell, the NADH formed by this reaction must be reoxidized to NAD+ for glycolysis to continue. Two major mechanisms for oxidizing NADH are: 1) the NADH-linked conversion of pyruvate to lactate (anaerobic). 2) oxidation of NADH via the respiratory chain (aerobic). Figure 23: Energy generating phase: conversion of glyceraldehyd e 3-phosphate to pyruvate. G. Synthesis of 3-phosphoglycerate producing ATP  When 1,3-BPG is converted to 3-phosphoglycerate, the high- energy phosphate group of 1,3-BPG is used to synthesize ATP from ADP (see Figure 23).  This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible.  Because two molecules of 1,3-BPG are formed from each glucose molecule, this kinase reaction replaces the two ATP molecules consumed by the earlier formation of glucose 6-phosphate and fructose 1,6-bisphosphate. H. Shift of the phosphate group from carbon 3 to carbon 2  The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible (see Figure 23). I. Dehydration of 2-phosphoglycerate  The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the 2-phosphoglycerate molecule, resulting in the formation of phosphoenolpyruvate (PEP), which contains a high energy enol phosphate (see Figure 23).  The reaction is reversible despite the high-energy nature of the product. J. Formation of pyruvate producing ATP  The conversion of PEP to pyruvate is catalyzed by pyruvate kinase, the third irreversible reaction of glycolysis.  The equilibrium of the pyruvate kinase reaction favors the formation of ATP (see Figure 23).  [Note: This is another example of substrate-level phosphorylation.] 1) Feed-forward regulation: In liver, pyruvate kinase is activated by fructose 1,6- bisphosphate, the product of the phosphofructokinase reaction.  This feed-forward regulation has the effect of linking the two kinase activities: increased phosphofructokinase activity results in elevated levels of fructose 1,6-bisphosphate, which activates pyruvate kinase. 2) Covalent modulation of pyruvate kinase: * Phosphorylation by a cAMP- dependent protein kinase leads to inactivation of pyruvate kinase in the liver (Figure 24). * When blood glucose levels are low, elevated glucagon increases the intracellular level of cAMP, which causes the phosphorylation and inactivation of pyruvate kinase. * Therefore, PEP is unable to continue Figure 24: Covalent modification in glycolysis, but instead enters the of hepatic pyruvate kinase gluconeogenesis pathway. results in inactivation of enzyme. 3) Pyruvate kinase deficiency:  The normal, mature erythrocyte lacks mitochondria and is, therefore, completely dependent on glycolysis for production of ATP.  This high-energy compound is required to meet the metabolic needs of the red blood cell, and also to fuel the pumps necessary for the maintenance of the biconcave, flexible shape of the cell, which allows it to squeeze through narrow capillaries.  The anemia observed in glycolytic enzyme deficiencies is a consequence of the reduced rate of glycolysis, leading to decreased ATP production.  The resulting alterations in the red blood cell membrane lead to changes in the shape of the cell.  The premature death and lysis of red blood cells results in hemolytic anemia. K. Reduction of pyruvate to lactate  Lactate, formed by the action of lactate dehydrogenase, is the final product of anaerobic glycolysis in eukaryotic cells (Figure 25).  The formation of lactate is the major fate for pyruvate in lens and cornea of the eye, kidney medulla, testes, leukocytes and red blood cells, because these are all poorly vascularized and/or lack mitochondria. Figure 25: Interconversion of pyruvate and lactate. 1) Lactate formation in muscle:  In exercising skeletal muscle, NADH production (by glyceraldehyde 3- phosphate dehydrogenase and by the three NAD+-linked dehydrogenases of the citric acid cycle, exceeds the oxidative capacity of the respiratory chain. This results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate.  Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps.  Much of this lactate eventually diffuses into the bloodstream, and can be used by the liver to make glucose. 2) Lactate consumption:  The direction of the lactate dehydrogenase reaction depends on the relative intracellular concentrations of pyruvate and lactate, and on the ratio of NADH/NAD+ in the cell.  For example, in liver and heart, the ratio of NADH/NAD+ is lower than in exercising muscle. These tissues oxidize lactate to pyruvate. In the liver, pyruvate is either converted to glucose by gluconeogenesis or oxidized in the TCA cycle.  Heart muscle exclusively oxidizes lactate to CO2 and H2O via the citric acid cycle. 3) Lactic acidosis:  Elevated concentrations of lactate in the plasma, termed lactic acidosis, occur when there is a collapse of the circulatory system, such as in myocardial infarction, pulmonary embolism, and uncontrolled hemorrhage, or when an individual is in shock.  The failure to bring adequate amounts of oxygen to the tissues results in impaired oxidative phosphorylation and decreased ATP synthesis.  To survive, the cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the end product. L. Energy yield from glycolysis  Despite the production of some ATP during glycolysis, the end products, pyruvate or lactate, still contain most of the energy originally contained in glucose. The TCA cycle is required to release that energy completely. 1) Anaerobic glycolysis: Two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate. There is no net production or consumption of NADH. 2) Aerobic glycolysis: The direct consumption and formation of ATP is the same as in anaerobic glycolysis that is, a net gain of two ATP per molecule of glucose. Two molecules of NADH are also produced per molecule of glucose. *Ongoing aerobic glycolysis requires the oxidation of most of this NADH by the electron transport chain, producing approximately three ATP for each NADH molecule entering the chain. [Note: NADH cannot cross the inner mitochondrial membrane, and substrate shuttles are required.]  VI. HORMONAL REGULATION OF GLYCOLYSIS  Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver (Figure 26).  These changes reflect an increase in gene transcription, resulting in increased enzyme synthesis.  High activity of these three enzymes favors the conversion of glucose to pyruvate, a characteristic of the well-fed state.  Conversely, gene transcription and synthesis of glucokinase, phosphofructokinase, and pyruvate kinase are decreased when plasma glucagon is high and insulin is low, for example, as seen in fasting or diabetes. Figure 26:  Effect of insulin and glucagon on the synthesis of key enzymes of glycolysis in liver.  VII. ALTERNATE FATES OF PYRUVATEA. A. Oxidative decarboxylation of pyruvate  Oxidative decarboxylation of pyruvate by pyruvate dehydrogenase complex is an important pathway in tissues with a high oxidative capacity, such as cardiac muscle.  Pyruvate dehydrogenase irreversibly converts pyruvate, the end product of glycolysis, into acetyl CoA, a major fuel for the TCA cycle and the building block for fatty acid synthesis. B. Carboxylation of pyruvate to oxaloacetate  Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is a biotin-dependent reaction. This reaction is important because it replenishes the citric acid cycle intermediates, and provides substrate for gluconeogenesis. C. Reduction of pyruvate to ethanol (microorganisms)  The decarboxylation of pyruvate by pyruvate decarboxylase occurs in yeast and other microorganisms, but not in humans. Figure 27:  Summary of the metabolic fates of pyruvate. ‫اﻟﻤﺤﺎﺿﺮة اﻟﺨﺎﻣﺴﺔ‬  TRICARBOXYLIC ACID CYCLE I. OVERVIEW  The tricarboxylic acid cycle (TCA cycle, also called the Krebs cycle or the citric acid cycle) plays several roles in metabolism.  It is the final pathway where the oxidative metabolism of carbohydrates, amino acids, and fatty acids converge, their carbon skeletons being converted to CO2.  This oxidation provides energy for the production of the majority of ATP in most animals, including humans.  The cycle occurs totally in the mitochondria and is, therefore, in close proximity to the reactions of electron transport , which oxidize the reduced coenzymes produced by the cycle.  The TCA cycle is an aerobic pathway, because O2 is required as the final electron acceptor.  Most of the body's catabolic pathways converge on the TCA cycle. Reactions such as the catabolism of some amino acids generate intermediates of the cycle and are called anaplerotic reactions. II. REACTIONS OF THE TCA CYCLE * In the TCA cycle, oxaloacetate is first condensed with an acetyl group from acetyl coenzyme A (CoA), and then is regenerated as the cycle is completed (Figure 28). *Thus, the entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates. [Note: Two carbons entering the Figure 28: The tricarboxylic acid cycle as acetyl CoA are balanced cycle shown as a part of the by two CO2 exiting.] central pathways of energy metabolism. A. Oxidative decarboxylation of pyruvate  Pyruvate, the endproduct of aerobic glycolysis, must be transported into the mitochondrion before it can enter the TCA cycle.  This is accomplished by a specific pyruvate transporter that helps pyruvate cross the inner mitochondrial membrane.  Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex, which is a multienzyme complex.  the pyruvate dehydrogenase complex is not part of the TCA cycle proper, but is a major source of acetyl CoA—the two-carbon substrate for the cycle. 1. Component enzymes: *The pyruvate dehydrogenase complex (PDH complex) is a multimolecular aggregate of three enzymes, pyruvate dehydrogenase (PDH or E1, also called a decarboxylase), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). * Each catalyzes a part of the overall reaction (Figure 29). *Their physical association links the reactions in proper sequence without the release of intermediates. *In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound regulatory enzymes, pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. 2. Coenzymes:  *The PDH complex contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions shown in Figure 29. E1 requires thiamine pyrophosphate (TPP), E2 requires lipoic acid and CoA, and E3 requires FAD and NAD+. Figure 29: Mechanism of action of the pyruvate dehydrogenase complex. TPP = thiamine pyrophosphate; L = lipoic acid.  3. Regulation of the pyruvate dehydrogenase complex: Figure 30: Regulation of pyruvate dehydrogenase complex. [ ……. denotes product inhibition.] 4. Pyruvate dehydrogenase deficiency: A deficiency in the E1 component of the PDH complex, although rare, is the most common biochemical cause of congenital lactic acidosis.  This enzyme deficiency results in an inability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactic acid via lactate dehydrogenase.  This causes particular problems for the brain, which relies on the TCA cycle for most of its energy, and is particularly sensitive to acidosis.  Symptoms are variable and include neurodegeneration, muscle spasticity and, in the neonatal onset form, early death.  it affects both males and females.  There is no proven treatment for pyruvate dehydrogenase deficiency: however, dietary restriction of carbohydrate and supplementation with TPP may reduce symptoms in select patients. B. Synthesis of citrate from acetyl CoA and oxaloacetate:  The condensation of acetyl CoA and oxaloacetate to form citrate (a tricarboxylic acid) is catalyzed by citrate synthase (Figure 31).  This aldol condensation has an equilibrium far in the direction of citrate synthesis.  In humans, citrate synthase is not an allosteric enzyme. It is inhibited by its product, citrate.  Note: Citrate, in addition to being an intermediate in the TCA cycle, provides a source of acetyl CoA for the cytosolic synthesis of fatty acids.  Citrate also inhibits phosphofructokinase, the rate-limiting enzyme of glycolysis , and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis. Figure 31: Formation of α-keto glutarate from acetyl CoA and oxaloacetate. C. Isomerization of citrate  Citrate is isomerized to isocitrate by aconitase, an Fe-S protein (see Figure 31).  [Note: Aconitase is inhibited by fluoroacetate, a compound that is used as a rat poison. Fluoroacetate is converted to fluoro acetyl CoA, which condenses wit oxaloacetate to form fluoro citrate—a potent inhibitor of aconitase—resulting in citrate accumulation.] D. Oxidation and decarboxylation of isocitrate  Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate, yielding the first of three NADH molecules produced by the cycle, and the first release of CO2 (see Figure 31).  This is one of the rate-limiting steps of the TCA cycle.  The enzyme is allosterically activated by ADP (a low-energy signal) and Ca2+, and is inhibited by ATP and NADH, whose levels are elevated when the cell has abundant energy stores. E. Oxidative decarboxylation of α- ketoglutarate  The conversion of α-ketoglutarate to succinyl CoA is catalyzed by the α- ketoglutarate dehydrogenase complex, a multimolecular aggregate of three enzymes (Figure 32).  The mechanism of this oxidative decarboxylation is very similar to that used for the conversion of pyruvate to acetyl CoA by the PDH complex.  The reaction releases the second CO2 and produces the second NADH of the cycle.  The coenzymes required are thiamine pyrophosphate, lipoic acid, FAD, NAD+, and CoA. F. Cleavage of succinyl CoA  Succinate thiokinase (also called succinyl CoA synthetase—named for the reverse reaction) cleaves the high-energy thioester bond of succinyl CoA.  This reaction is coupled to phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP).  GTP and ATP are energetically inter convertible by the nucleo side diphosphate kinase reaction: GTP + ADP GDP + ATP  The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation. G. Oxidation of succinate  Succinate is oxidized to fumarate by succinate dehydrogenase, as FAD (its coenzyme) is reduced to FADH2.  Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane.  [Note: FAD, rather than NAD+, is the electron acceptor because the reducing power of succinate is not sufficient to reduce NAD+.] H. Hydration of fumarate  Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (also called fumarate hydratase.  [Note: Fumarate is also produced by the urea cycle, in purine synthesis, and during catabolism of the amino acids, phenylalanine and tyrosine. Figure 32: Formation of malate from α- ketoglutarate. I. Oxidation of malate  Malate is oxidized to oxaloacetate by malate dehydrogenase.  This reaction produces the third and final NADH of the cycle.  The ΔG0 of the reaction is positive, but the reaction is driven in the direction of oxaloacetate by the highly exergonic citrate synthase reaction.  [Note: Oxaloacetate is also produced by the transamination of the amino acid, aspartic acid  III. ENERGY PRODUCED BY THE TCA CYCLE  Two carbon atoms enter the cycle as acetyl CoA and leave as CO2.  The cycle does not involve net consumption or production of oxaloacetate or of any other intermediate.  Four pairs of electrons are transferred during one turn of the cycle: three pairs of electrons reducing three NAD+ to NADH and one pair reducing FAD to FADH2.  Oxidation of one NADH by the electron transport chain leads to formation of approximately three ATP, whereas oxidation of FADH2 yields approximately two ATP.  The total yield of ATP from the oxidation of one acetyl CoA is shown in below Figure.  IV. REGULATION OF THE TCA CYCLE  In contrast to glycolysis, which is regulated primarily by phosphofructokinase, the TCA cycle is controlled by the regulation of several enzyme activities (see Figure 33).  The most important of these regulated enzymes are those that catalyze reactions with highly negative ΔG0: citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase complex. *Reducing equivalents needed for oxidative phosphorylation are generated by the pyruvate dehydrogenase complex and the TCA cycle, and both processes are up regulated in response to a rise in ADP. Figure 33: A. Production of reduced coenzymes, ATP, and CO2 in the citric acid cycle. Figure 33: B. Inhibitors and activators of the cycle.  Gluconeogenesis Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for only 10–18 hours in the absence of dietary intake of carbohydrate. During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is formed from precursors such as lactate, pyruvate, glycerol (derived from the backbone of triacylglycerols, and α-ketoacids (derived from the catabolism of glucogenic amino acids). The formation of glucose does not occur by a simple reversal of glycolysis, because the overall equilibrium of glycolysis strongly favors pyruvate formation. Instead, glucose is synthesized by a special pathway, gluconeogenesis, that requires both mitochondrial and cytosolic enzymes. During an overnight fast, approximately 90% of gluconeogenesis occurs in the liver, with the kidneys providing 10% of the newly synthesized glucose molecules. However, during prolonged fasting, the kidneys become major glucose-producing organs, contributing an estimated 40% of the total glucose production.