Glycolysis

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 gen- eration by degradation of complex molecules occurs in three stages as shown in Figure 8.3. [Note: Catabolic pathways are typically oxidative, and require coenzymes such as NAD+.]

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-bis- phosphate 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.

L. Energy yield from glycolysis

Despite the production of some ATP during glycolysis, the end prod- ucts, pyruvate or lactate, still contain most of the energy originally contained in glucose. The TCA cycle is required to release that energy completely

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 con- centration 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.

Anabolic pathways

Anabolic reactions combine small molecules, such as amino acids, to form complex molecules, such as proteins (Figure 8.4). Anabolic reac- tions 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 elec- tron donor NADPH (see p. 147). 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 pro- cess in which a few biosynthetic precursors form a wide variety of polymeric or complex products.

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. [Note: Aldolase B, the iso- form in the liver and kidney, also cleaves fructose 1-phosphate, and functions in the metabolism of dietary fructose

Three stages of catabolism.

1. Hydrolysis of complex molecules 2. Conversion of building blocks to simple intermediates 3. Oxidation of acetyl CoA

1. Regulation by energy levels within the cell

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 alloster- ically by high concentrations of AMP, which signal that the cell's energy stores are depleted. [Note: Citrate inhibition favors the use of glucose for glycogen synthesis,

ALTERNATE FATES OF PYRUVATE

A. Oxidative decarboxylation of pyruvate B. Carboxylation of pyruvate to oxaloacetate C. Reduction of pyruvate to ethanol (microorganisms)

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 inter- mediates, and provides substrate for gluconeogenesis

3. Lactic acidosis:

Elevated concentrations of lactate in the plasma, termed lactic acidosis, occur when there is a collapse of the circu- latory 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 lac- tic acid as the endproduct. [Note: 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 oxy- gen required to recover from a period when the availability of oxy- gen has been inadequate is termed the oxygen debt. The oxygen debt is often related to patient mor- bidity 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.

b. 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-bisphos- phate. This results in a decrease in the overall rate of glycolysis and an increase in gluconeogenesis.

2. Regulation by fructose 2,6-bisphosphate:

Fructose 2,6-bisphos- phate 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 dephosphory- lates fructose 2,6-bisphosphate back to fructose 6-phosphate. In liver, the kinase domain is active if dephosphorylated and is inac- tive if phosphorylated (Figure 8.17). [Note: 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-bisphos- phate 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.]

b. Regulation by fructose 6-phosphate and glucose:

Glucokinase activity is not directly inhibited by glucose 6-phos- phate 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 GK 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. [Note: Fructose 1-phosphate inhibits formation of the glucokinase-GKRP complex.] Glucokinase functions as a glucose sensor in the maintenance of blood glucose homeosta- sis. Mutations that decrease the activity of glu- cokinase are the cause of a rare form of dia- betes, maturity onset diabetes of the young type 2 (MODY 2)

a. Kinetics:

Glucokinase differs from hexokinase in several impor- tant 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 carbohy- drate-rich meal, when high levels of glucose are delivered to the liver via the portal vein. Glucokinase has a high Vmax, allow- ing 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. [Note: GLUT-2 insures that blood glucose equilibrates rapidly across the membrane of the hepatocyte.]

TRANSPORT OF GLUCOSE INTO CELLS

Glucose cannot diffuse directly into cells, but enters by one of two trans- port mechanisms: a Na+-independent, facilitated diffusion transport sys- tem or a Na+-monosaccharide cotransporter system.

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 chemi- cal 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. "Second messenger" molecules—so named because they intervene between the original messenger (the neurotransmitter or hormone) and the ultimate effect on the cell—are part of the cascade of events that translates hormone or neurotransmitter binding into a cellular response. Two of the most widely recognized second messenger systems are the calcium/phosphatidylinositol system (see p. 205), and the adenylyl cyclase system, which is particularly important in regulating the pathways of intermediary metabolism.

INTRODUCTION TO METABOLISM

In Chapter 5, individual enzymic reactions were analyzed in an effort to explain the mechanisms of catalysis. However, in cells, these reactions rarely occur in isolation, but rather are organized into multistep sequences called pathways, such as that of glycolysis (Figure 8.1). In a pathway, the product of one reaction serves as the substrate of the sub- sequent reaction. Different pathways can also intersect, forming an inte- grated and purposeful network of chemical reactions. These are collectively called metabolism, which is the sum of all the chemical 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. [Note: Pathways that regener- ate a component are called cycles.] In the following chapters, this text focuses on the central metabolic pathways that are involved in synthe- sizing and degrading carbohydrates, lipids, and amino acids.

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 exer- cise, lactate accumulates in muscle, causing a drop in the intra- cellular 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. 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. For example, GLUT-1, GLUT-3, and GLUT-4 are primarily involved in glucose uptake from the blood. In contrast, 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). [Note: GLUT-2 is also found in pancreatic β cells.] GLUT-5 is unusual in that it is the primary transporter for fructose (instead of glucose) in the small intestine and the testes.

2. Glucokinase

In liver parenchymal cells and β cells of the pan- creas, glucokinase (also called hexokinase D, or type IV) is the predominant enzyme responsible for the phosphorylation of glu- cose. In β 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 hyper- glycemia. [Note: 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.

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.

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 phos- phorylate 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 affin- ity, see p. 59) for glucose. This permits the efficient phosphoryla- tion and subsequent metabolism of glucose even when tissue concentrations of glucose are low (Figure 8.13). Hexokinase, however, 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.

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, poly- saccharides 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 com- pared with the energy produced during the third stage of catabolism.

Metabolic map

It is convenient to investigate metabolism by examining its compo- nent pathways. Each pathway is composed of multienzyme sequences, and each enzyme, in turn, may exhibit important catalytic or regulatory features. To provide the reader with the "big picture," a metabolic map containing the important central pathways of energy metabolism is presented in Figure 8.2. This map is useful in tracing connections between pathways, visualizing the purposeful "move- ment" 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. Throughout the next three units of this book, each pathway under discussion will be repeatedly featured as part of the major metabolic map shown in Figure 8.2.

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.

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 dehydro- genase 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

A. Phosphorylation of glucose

Phosphorylated sugar molecules do not readily penetrate cell mem- branes, 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 glu- cose (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

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 glycoly- sis, 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.

Communication between cells (intercellular)

The ability to respond to extracellular signals is essential for the sur- vival 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 for- mation 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.

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 pyru- vate kinase reaction favors the formation of ATP (see Figure 8.18). [Note: This is another example of substrate-level phosphorylation.]

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.

F. Oxidation of glyceraldehyde 3-phosphate

The conversion of glyceraldehyde 3-phosphate to 1,3-bisphospho- glycerate by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis (Figure 8.18). [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 gly- colysis 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 (aero- bic, see p. 75). The latter requires substrate shuttles

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 micro- organisms, but not in humans. The enzyme requires thiamine pyro- phosphate as a coenzyme, and catalyzes a reaction similar to that described for pyruvate dehydrogenase

I. Dehydration of 2-phosphoglycerate

The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the 2-phosphoglycerate molecule, resulting in the for- mation 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.

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 trans- port chain, producing approximately three ATP for each NADH molecule entering the chain (see p. 77). [Note: NADH cannot cross the inner mitochondrial membrane, and substrate shuttles are required

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 con- verted to glucose by gluconeogenesis or oxidized in the TCA cycle. Heart muscle exclusively oxidizes lactate to CO2 and H2O via the citric acid cycle.

1. 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 sub- units) of the cell membrane. These proteins, referred to as G pro- teins 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 dis- sociates from the βγ subunits and moves to adenylyl cyclase, which is thereby activated. Many molecules of active Gα protein are formed by one activated receptor. [Note: The ability of a hor- mone 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 (not shown in Figure 8.7).] 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 βγ 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.

1. Tissue specificity of GLUT gene expression:

The glucose trans- porters display a tissue-specific pattern of expression. For exam- ple, 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 tis- sue and skeletal muscle. [Note: The number of GLUT-4 trans- porters active in these tissues is increased by insulin. (See p. 311 for a discussion of insulin and glucose transport.)] The other GLUT isoforms also have tissue-specific distributions.

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.

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 8.16). PFK-1 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate, and by regulatory substances described below.

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 reac- tion is readily reversible and is not a rate-limiting or regulated step.

2. Protein kinases:

The next key link in the cAMP second messen- ger 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. [Note: Several types of protein kinases are not cAMP-dependent, for example, protein kinase C

3. Pyruvate kinase deficiency

The normal, mature erythrocyte lacks mitochondria and is, therefore, completely dependent on glycoly- sis 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 deficien- cies 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 (gen- erally 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 defi- ciency have a mutant enzyme that shows abnormal properties— most often altered kinetics Pyruvate kinase deficiency is the second most common cause (after glucose 6-phosphate dehy- drogenase deficiency) of enzyme deficiency- related nonspherocytic hemolytic anemia.

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 con- serves 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.

REGULATION OF METABOLISM

The pathways of metabolism must be coordinated so that the produc- tion 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 sophisticated 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

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.

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 inhibi- tion, 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.

Adenylyl cyclase

The recognition of a chemical signal by some membrane receptors, such as the β- and α2-adrenergic receptors, triggers either an increase or a decrease in the activity of adenylyl cyclase (adenylate cyclase). This is a membrane-bound enzyme that 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-bind- ing region, seven transmembrane helices, and an intracellular domain that interacts with G proteins

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 min- utes 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, Regular consumption of meals rich in carbo- hydrate 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.

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 phos- phoglycerate by phosphoglycerate mutase is freely reversible (see Figure 8.18).

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 inor- ganic 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 gly- colytic pathway. [Note: Arsenic also replaces Pi on the F1 domain of ATP synthase (see p. 78), resulting in formation of ADP-arsen- ate that is rapidly hydrolyzed.]

3. Oxidation of acetyl CoA

The tricarboxylic acid (TCA) cycle (see p. 109) is the final common pathway in the oxidation of fuel molecules that produce acetyl CoA. Oxidation of acetyl CoA gen- erates large amounts of ATP via oxidative phosphorylation as electrons flow from NADH and FADH2 to oxygen

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 con- sumed by the earlier formation of glucose 6-phosphate and fructose 1,6-bisphosphate. [Note: This is an example of substrate-level phos- phorylation, 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).]

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-depen- dent-glucose transporter or SGLT. This type of transport occurs in the epithelial cells of the intestine (see p. 87), renal tubules, and choroid plexus. [Note: The choroid plexus, part of the blood brain barrier, also contains GLUT-1.]

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 mem- brane in two conformational states (Figure 8.10). Extracellular glu- cose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane.

E. Isomerization of dihydroxyacetone phosphate

Triose phosphate isomerase interconverts dihydroxyacetone phos- phate and glyceraldehyde 3-phosphate (see Figure 8.16). 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 glycer- aldehyde 3-phosphate from the cleavage products of fructose 1,6- bisphosphate.

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.

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. [Note: Phosphodiesterase is inhibited by methylxanthine derivatives, such as theophylline and caffeine.1]