Glycolysis PDF

# Glycolysis ## I. Introduction to Metabolism - In Chapter 5, individual enzymic reactions were analyzed. - In cells, these reactions rarely occur in isolation, but are organized into multistep sequences called **pathways**. - An example is **glycolysis** (Figure 8.1). - In a pathway, the product...

# Glycolysis ## I. Introduction to Metabolism - In Chapter 5, individual enzymic reactions were analyzed. - In cells, these reactions rarely occur in isolation, but are organized into multistep sequences called **pathways**. - An example is **glycolysis** (Figure 8.1). - In a pathway, the product of one reaction serves as the substrate of the subsequent reaction. - Different pathways can also interconnect to form an integrated network of chemical reactions, collectively called **metabolism**. - Metabolism is the sum of chemical changes occurring in a cell, tissue, or the whole body. - Most metabolic pathways can be classified as either **catabolic** (degradative) or **anabolic** (synthetic). - Catabolic reactions break down complex molecules, such as proteins, polysaccharides, and lipids, into simpler molecules like CO2, NH3 (ammonia), and water. - Anabolic pathways form complex end products from simple precursors, such as the synthesis of the polysaccharide glycogen from glucose. - Pathways that regenerate a component are called **cycles**. - The following chapters focus on central metabolic pathways, which are involved in synthesizing and degrading carbohydrates, lipids, and amino acids. ## A. Metabolic Map To better understand metabolism, it can be visualized by examining its component pathways: - Each pathway is composed of multienzyme sequences. - Each enzyme has a unique catalytic or regulatory feature. - **Metabolic maps** (Figure 8.2) visually present the central pathways of energy mechanisms. - These maps are helpful in tracing connections between pathways, visualizing the "movement" of metabolic intermediates, and picturing the effect on the flow of intermediates if a pathway is blocked, for example, by a drug or an inherited deficiency of an enzyme. - Each pathway under discussion in this book will be repeatedly featured in Figure 8.2 to provide the major metabolic map. ## B. Catabolic Pathways - Catabolic reactions serve to capture chemical energy in the form of adenosine triphosphate (ATP) from the degradation of energy-rich fuel molecules. - These fuel molecules can originate from the diet or be stored in cells. - Catabolism also allows molecules in the diet to be converted into building blocks for the synthesis of complex molecules. - Energy generation by degradation of complex molecules generally occurs in three stages (Figure 8.3): - **Hydrolysis of complex molecules**: complex molecules are broken down into their component building blocks. Examples include the degradation of proteins to amino acids, polysaccharides to monosaccharides, and fats (triacylglycerols) to free fatty acids and glycerol. - **Conversion of building blocks to simple intermediates**: building blocks are further degraded to acetyl coenzyme A (CoA) and a few other simple molecules. Some energy is captured as ATP during this stage, but the amount produced is small compared to the third stage. - **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. ## C. Anabolic Pathways - Anabolic reactions combine small molecules, such as amino acids, to form complex molecules, such as proteins (Figure 8.4). - These reactions require energy (are endergonic), 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. - Catabolism is a **convergent** process, transforming a wide variety of molecules into a few common end products. - In contrast, anabolism is a **divergent** process, where a few biosynthetic precursors form a wide variety of polymeric or complex products. ## I. 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. Moreover, individual cells do not function in isolation but, rather, are part of a community of interacting tissues. A sophisticated communication system has evolved to coordinate the functions of the body. ## A. Signals from Within the Cell (Intracellular) The rate of a metabolic pathway can respond to regulatory signals that arise from within the cell. - This regulation 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 responses generated by internal signals. - Cell communication can occur via surface-to-surface contact or by the formation of gap junctions, allowing direct communication between the cytoplasms of adjacent cells. - However, the most important route of communication for energy metabolism is chemical signaling between cells by bloodborne hormones or by neurotransmitters. ## C. Second Messenger Systems Hormones or neurotransmitters can be thought of as signals, and their receptors as signal detectors. - Each component serves as a link in the communication between extracellular events and chemical changes within the cell. - Many receptors signal their recognition of a bound ligand by initiating a series of reactions that ultimately result in a specific intracellular response. - These intermediary molecules that intervene between the original messenger (the neurotransmitter or hormone) and the ultimate effect on the cell, are called **second messengers**. - Two of the most widely recognized second messenger systems are the calcium/phosphatidylinositol system and the adenylyl cyclase system, which is particularly important in regulating the pathways of intermediary metabolism. ## D. Adenylyl Cyclase - The recognition of a chemical signal by some membrane receptors, such as the ẞ- and 2-adrenergic receptors, triggers either an increase or a decrease in the activity of adenylyl cyclase. - This membrane-bound enzyme converts ATP to 3',5'-adenosine monophosphate (also called cyclic AMP or cAMP). - The chemical signals are most often hormones or neurotransmitters, each of which binds to a unique type of membrane receptor. - Therefore, tissues that respond to more than one chemical signal must have several different receptors, each of which can be linked to adenylyl cyclase. - These receptors, known as **G protein-coupled receptors (GPCR)**, are characterized by an extracellular ligand-binding region, seven transmembrane helices, and an intracellular domain that interacts with G proteins (Figure 8.6). ## II. GTP-Dependent Regulatory Proteins - The effect of the activated, occupied GPCR on second messenger formation is not direct but, rather, is mediated by specialized trimeric proteins (α, β, γ subunits) of the cell membrane. - These proteins, referred to as **G proteins** because they bind guanosine nucleotides (GTP and GDP), form a link in the chain of communication between the receptor and adenylyl cyclase. - In the inactive form of a G protein, the α-subunit is bound to GDP (Figure 8.7). - Binding of ligand causes a conformational change in the receptor, triggering replacement of this GDP with GTP. - The GTP-bound form of the α subunit dissociates from the ẞy subunits and moves to adenylyl cyclase, which is thereby activated. - Many molecules of active Gα protein are formed by one activated receptor. - The ability of a hormone or neurotransmitter to stimulate or inhibit adenylyl cyclase depends on the type of Gα protein that is linked to the receptor. - One family of G proteins, designated Gs, stimulates adenylyl cyclase; - Another family, designated Gi, inhibits the enzyme. - The actions of the Gα-GTP complex are short-lived because Gα has an inherent GTPase activity, resulting in the rapid hydrolysis of GTP to GDP. This causes inactivation of the Gα, its dissociation from adenylyl cyclase and reassociation with the ẞy dimer. - Toxins from Vibrio cholerae (cholera) and Bordetella pertussis (whooping cough) cause inappropriate activation of adenylyl cyclase through covalent modification (ADP-ribosylation) of different G proteins. - With cholera, the GTPase activity of Gαs is inhibited. - With whooping cough, Gαi is inactivated. ## 2. Protein Kinases - The next key link in the cAMP second-messenger system is the activation by cAMP of a family of enzymes called **cAMP-independent protein kinases**, for example, protein kinase A (Figure 8.8). - Cyclic AMP activates protein kinase A by binding to its two regulatory subunits, causing the release of active catalytic subunits. - The active subunits catalyze the transfer of phosphate from ATP to specific serine or threonine residues of protein substrates. - The phosphorylated proteins may act directly on the cell's ion channels, or, if enzymes, may become activated or inhibited. - Protein kinase A can also phosphorylate proteins that bind to DNA, causing changes in gene expression. - Several types of protein kinases are not cAMP-dependent, for example, protein kinase C. ## 3. Dephosphorylation of Proteins - The phosphate groups added to proteins by protein kinases are removed by **protein phosphatases** - enzymes that hydrolytically cleave phosphate esters (see Figure 8.8). - This ensures that changes in protein activity induced by phosphorylation are not permanent. ## 4. Hydrolysis of cAMP - cAMP is rapidly hydrolyzed to 5'-AMP by **cAMP phosphodiesterase**, one of a family of enzymes that cleave the cyclic 3',5'-phosphodiester bond. - 5'-AMP is not an intracellular signaling molecule. - Thus, the effects of neurotransmitter- or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed. - Phosphodiesterase is inhibited by methylxanthine derivatives, such as theophylline and caffeine. ## 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 8.9A). - 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 8.9B). - 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 8.9C). - 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. ## 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 - a Na+-monosaccharide cotransporter system. ## A. Na+-Independent Facilitated Diffusion Transport - This system is mediated by a family of 14 glucose transporters in cell membranes. - These are designated GLUT-1 to GLUT-14 (glucose transporter isoforms 1–14). - These transporters exist in the membrane in two conformational states (Figure 8.10). - Extracellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane. ### 1. Tissue Specificity of GLUT Gene Expression - The glucose transporters display a tissue-specific pattern of expression. - GLUT-3 is the primary glucose transporter in neurons. - GLUT-1 is abundant in erythrocytes and blood brain barrier, but is low in adult muscle, whereas GLUT-4 is abundant in adipose tissue and skeletal muscle. - The number of GLUT-4 transporters active in these tissues is increased by insulin. - The other GLUT isoforms also have tissue-specific distributions. ### 2. Specialized Functions of GLUT Isoforms - In facilitated diffusion, glucose movement follows a concentration gradient, that is, from a high glucose concentration to a lower one. - GLUT-1, GLUT-3, and GLUT-4 are primarily involved in glucose uptake from the blood. - GLUT-2, which is found in the liver and kidney, can either transport glucose into these cells when blood glucose levels are high, or transport glucose from these cells when blood glucose levels are low (for example, during fasting). - GLUT-5 is unusual in that it is the primary transporter for fructose (instead of glucose) in the small intestine and the testes. ## 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. - The choroid plexus, part of the blood brain barrier, also contains GLUT-1. ## V. Reactions of Glycolysis - The conversion of glucose to pyruvate occurs in two stages (Figure 8.11). - 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 (see p. 102) per glucose molecule metabolized. ## 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 8.12), 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-phosphate. ### 1. Hexokinase - In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three regulatory enzymes of glycolysis (see also phosphofructokinase and pyruvate kinase). - 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 (and, therefore, a high affinity, see p. 59) for glucose. - This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low (Figure 8.13). - However, hexokinase has a low Vmax for glucose and, therefore, cannot sequester (trap) cellular phosphate in the form of phosphorylated hexoses, or phosphorylate more sugars than the cell can use. ### 2. Glucokinase - In liver parenchymal cells and ẞ cells of the pancreas, glucokinase (also called hexokinase D, or type IV) is the predominant enzyme responsible for the phosphorylation of glucose. - In ẞ cells, glucokinase functions as the glucose sensor, determining the threshold for insulin secretion (see p. 310). - In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia. - Hexokinase also serves as a glucose sensor in neurons of the hypothalamus, playing a key role in the adrenergic response to hypoglycemia (see p. 315). - Despite the popular but misleading name glucokinase, the sugar specificity of the enzyme is similar to that of other hexokinase isozymes. #### a. Kinetics - Glucokinase differs from hexokinase in several important properties. - For example, it has a much higher Km, requiring a higher glucose concentration for half-saturation (see Figure 8.13). - Thus, 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. - GLUT-2 insures that blood glucose equilibrates rapidly across the membrane of the hepatocyte. #### b. Regulation by Fructose 6-Phosphate and Glucose - Glucokinase activity is not directly inhibited by glucose 6-phosphate as are the other hexokinases, but rather is indirectly inhibited by fructose 6-phosphate (which is in equilibrium with glucose 6-phosphate, a product of glucokinase), and is indirectly stimulated by glucose (a substrate of glucokinase) via the following mechanism. - Glucokinase regulatory protein (GKRP) in the liver regulates the activity of glucokinase through reversible binding. - In the presence of fructose 6-phosphate, glucokinase is translocated into the nucleus and binds tightly to the regulatory protein, thus rendering the enzyme inactive (Figure 8.14). - When glucose levels in the blood (and also in the hepatocyte, as a result of GLUT-2) increase, glucokinase is released from the regulatory protein, and the enzyme re-enters the cytosol where it phosphorylates glucose to glucose 6-phosphate. - Fructose 1-phosphate inhibits formation of the glucokinase-GKRP complex. - Glucokinase functions as a glucose sensor in the maintenance of blood glucose homeostasis. - Mutations that decrease the activity of glucokinase are the cause of a rare form of diabetes, maturity onset diabetes of the young type 2 (MODY 2). ## B. Isomerization of Glucose 6-Phosphate - The isomerization of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase (Figure 8.15). - The reaction is readily reversible and is not a rate-limiting or regulated step. ## C. Phosphorylation of Fructose 6-Phosphate - The irreversible phosphorylation reaction catalyzed by phosphofructokinase-1 (PFK-1) is the most important control point and the rate-limiting and committed step of glycolysis (Figure 8.16). - PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate, and by regulatory substances described below. ### 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 (see p. 109), also inhibit PFK-1. - Conversely, PFK-1 is activated allosterically by high concentrations of AMP, which signal that the cell's energy stores are depleted. - Citrate inhibition favors the use of glucose for glycogen synthesis, see p.125. ### 2. Regulation by Fructose 2,6-Bisphosphate - Fructose 2,6-bisphosphate is the most potent activator of PFK-1 (see Figure 8.16), 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 (Figure 8.17). - Fructose 2,6-bisphosphate is an inhibitor of fructose 1,6-bisphosphatase, an enzyme of gluconeogenesis (see p. 120 for a discussion of the regulation of gluconeogenesis). - The reciprocal actions of fructose 2,6-bisphosphate on glycolysis (activation) and gluconeogenesis (inhibition) ensure that both pathways are not fully active at the same time, preventing a futile cycle in which glucose would be converted to pyruvate followed by resynthesis of glucose from pyruvate. - 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 (see Figure 8.17). Fructose 2,6-bisphosphate, therefore, acts as an intracellular signal, indicating that glucose is abundant. - During starvation: Elevated levels of glucagon and low levels of insulin, such as occur during fasting (see p. 327), 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 dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 8.16). - The reaction is reversible and not regulated. - Aldolase B, the isoform in the liver and kidney, also cleaves fructose 1-phosphate, and functions in the metabolism of dietary fructose (see p. 138). ## E. Isomerization of Dihydroxyacetone Phosphate - Triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 8.16). - Dihydroxyacetone 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 8.18). - 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, see p. 96), and 2) oxidation of NADH via the respiratory chain (aerobic, see p. 75). - The latter requires substrate shuttles (see p. 79.) ### 1. Synthesis of 1,3-Bisphosphoglycerate (1,3-BPG) - The oxidation of the aldehyde group of glyceraldehyde 3-phosphate to a carboxyl group is coupled to the attachment of Pi to the carboxyl group. - The high-energy phosphate group at carbon 1 of 1,3-BPG conserves much of the free energy produced by the oxidation of glyceraldehyde 3-phosphate. - The energy of this high-energy phosphate drives the synthesis of ATP in the next reaction of glycolysis. ### 2. Mechanism of Arsenic Poisoning - The toxicity of arsenic is explained primarily by the inhibition of enzymes such as pyruvate dehydrogenase, which require lipoic acid as a coenzyme (see p. 110). - However, pentavalent arsenic (arsenate) also can prevent net ATP and NADH production by glycolysis, without inhibiting the pathway itself. - The poison does so by competing with inorganic phosphate as a substrate for glyceraldehyde 3-phosphate dehydrogenase, forming a complex that spontaneously hydrolyzes to form 3-phosphoglycerate (see Figure 8.18). - By bypassing the synthesis of and phosphate transfer from 1,3-BPG, the cell is deprived of energy usually obtained from the glycolytic pathway. - Arsenic also replaces P₁ on the F₁ domain of ATP synthase (see p. 78), resulting in formation of ADP-arsenate that is rapidly hydrolyzed. ### 3. Synthesis of 2,3-Bisphosphoglycerate (2,3-BPG) in Red Blood Cells - Some of the 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase (see Figure 8.18). - 2,3-BPG, which is found in only trace amounts in most cells, is present at high concentration in red blood cells (increases O2 delivery, see p. 31). - 2,3-BPG is hydrolyzed by a phosphatase to 3-phosphoglycerate, which is also an intermediate in glycolysis (see Figure 8.18). - In the red blood cell, glycolysis is modified by inclusion of these “shunt” reactions. ## 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 8.18). - 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. - This is an example of substrate-level phosphorylation, in which the energy needed for the production of a high-energy phosphate comes from a substrate rather than from the electron transport chain (see J. below and p. 113 for other examples). ## 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 8.18). ## 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 8.18). - 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 8.18). - 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 (instead of the more usual feedback) 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 8.19). - 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 in glycolysis, but instead enters the gluconeogenesis pathway. - This, in part, explains the observed inhibition of hepatic glycolysis and stimulation of gluconeogenesis by glucagon. - Dephosphorylation of pyruvate kinase by a phosphoprotein phosphatase results in reactivation of the 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 and, ultimately, to phagocytosis by the cells of the reticuloendothelial system, particularly macrophages of the spleen. - The premature death and lysis of red blood cells results in hemolytic anemia. - Among patients exhibiting the rare genetic defects of glycolytic enzymes, about 95% show a deficiency in pyruvate kinase, and 4% exhibit phosphoglucose isomerase deficiency. - PK deficiency is restricted to the erythrocytes, and produces mild to severe chronic hemolytic anemia (erythrocyte destruction), with the severe form requiring regular cell transfusions. - The severity of the disease depends both on the degree of enzyme deficiency (generally 5-25% of normal levels), and on the extent to which the individual's red blood cells compensate by synthesizing increased levels of 2,3-BPG (see p. 31). - Almost all individuals with PK deficiency have a mutant enzyme that shows abnormal properties-most often altered kinetics (Figure 8.20). - Pyruvate kinase deficiency is the second most common cause (after glucose 6-phosphate dehydrogenase deficiency) of enzyme deficiency-related nonspherocytic 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 8.21). - 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. ### 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, see p. 112) 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 (see p. 118). ### 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 (obtained from the blood) 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 endproduct. - Production of even meager amounts of ATP may be life-saving during the period required to reestablish adequate blood flow to the tissues. - The excess oxygen required to recover from a period when the availability of oxygen has been inadequate is termed the oxygen debt. - The oxygen debt is often related to patient morbidity or mortality. - In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of oxygen debt in patients and the monitoring of their recovery. ## 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 (see p. 109). ### 1. Anaerobic Glycolysis - Two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate (Figure 8.22). - 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 (see p. 77). - NADH cannot cross the inner mitochondrial membrane, and substrate shuttles are required (see p. 79). ## VI. Hormonal Regulation of Glycolysis - The regulation of glycolysis by allosteric activation or inhibition, or the phosphorylation/dephosphorylation of rate-limiting enzymes, is short-term-that is, they influence glucose consumption over periods of minutes or hours. - Superimposed on these moment-to-moment effects are slower, and often more profound, hormonal influences on the amount of enzyme protein synthesized. - These effects can result in 10-fold to 20-fold increases in enzyme activity that typically occur over hours to days. - Although the current focus is on glycolysis, reciprocal changes occur in the rate-limiting enzymes of gluconeogenesis, which are described in Chapter 10 (see p. 117). - 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 8.23). - 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 (see p. 321). - 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. ## VII. Alternate Fates of Pyruvate ### 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 (Figure 8.24). - Pyruvate dehydrogenase irreversibly converts pyruvate, the end product of glycolysis, into acetyl CoA, a major fuel for the TCA cycle (see p. 109) and the building block for fatty acid synthesis (see p. 183). ### B. Carboxylation of Pyruvate to Oxaloacetate - Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is a biotin-dependent reaction (see Figure 8.24). - This reaction is important because it replenishes the citric acid cycle intermediates, and provides substrate for gluconeogenesis (see p. 118). ### C. Reduction of Pyruvate to Ethanol (Microorganisms) - The conversion of pyruvate to ethanol occurs by the two reactions summarized in Figure 8.24. - The decarboxylation of pyruvate by pyruvate decarboxylase occurs in yeast and certain other microorganisms, but not in humans. - The enzyme requires thiamine pyrophosphate as a coenzyme, and catalyzes a reaction similar to that described for pyruvate dehydrogenase (see p. 110). ## VIII. Chapter Summary - Most pathways can be classified as either catabolic (degrade complex molecules to a few simple products) or anabolic (synthesize complex end products from simple precursors). - Catabolic reactions also capture chemical energy in the form of ATP from the degradation of energy-rich molecules. - Anabolic reactions require energy, which is generally provided by the breakdown of ATP. - The rate of a metabolic pathway can respond to regulatory signals, for example, allosteric activators or inhibitors, that arise from within the cell. - Signaling between cells provides for the integration of metabolism. - The most important route of this communication is chemical signaling between cells, for example, by hormones or neurotransmitters.