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الكلية كلية الطب
القسم الكيمياء الحياتية
المرحلة 2
أستاذ المادة عبد السميع حسن حمود الطائي
5/7/2011 8:47:40 PM
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 1). 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. Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the citric acid cycle. Alternatively, glucose can be converted to pyruvate, which is reduced by NADH to form lactate. This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of oxygen. Anaerobic glycolysis allows the continued production of ATP in tissues that lack mitochondria (for example, red blood cells) or in cells deprived of sufficient oxygen.
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 or a Na+-monosaccharide cotransporter system.
A. Na+-independent facilitated diffusion transport
This system is mediated by a family of at least fourteen glucose transporters in cell membranes. They are designated GLUT-1 to GLUT-14 (glucose transporter isoforms 1 to 14). These transporters exist in the membrane in two conformational states (Figure 2). 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. For example, GLUT-1 is abundant in erythrocytes and brain, but is low in adult muscle, GLUT-3 is the primary glucose transporter in neurons, whereas GLUT-4 is abundant in adipose tissue and skeletal muscle. [Note: 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, 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, kidney, and ? cells of the pancreas, can either transport glucose into these cells when blood glucose levels are high, or transport glucose from the cells to the blood 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 in the testes. GLUT-7, which is expressed in the liver and other gluconeogenic tissues, mediates glucose flux across the endoplasmic reticular membrane.
B. Na+-monosaccharide cotransporter system
This is an energy-requiring process that transports glucose "against" a concentration gradient, 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. This type of transport occurs in the epithelial cells of the intestine, renal tubules, and choroid plexus.
REACTIONS OF GLYCOLYSIS
The conversion of glucose to pyruvate occurs in two stages. 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. [Note: Two molecules of NADH are formed when pyruvate is produced (aerobic glycolysis), whereas NADH is reconverted to NAD+ when lactate is the end product (anaerobic 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 they are too polar to diffuse through the cell membrane. The irreversible phosphorylation of glucose (Figure 3), 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. 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. Hexokinase has a low Km (and, therefore, a high affinity) for glucose. This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue , concentrations of glucose are low (Figure 4). 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.
2. Glucokinase: In liver parenchymal cells and islet cells of the pancreas, glucokinase (also called hexokinase D, or type IV) is the predominant enzyme responsible for the phosphorylation of glucose. In ? cells, glucokinase functions as the glucose sensor, determining the threshold for insulin secretion. In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia. [Note: 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 4). 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.
b. Regulation by fructose 6-phosphate and glucose: Glucokinase activity is not allosterically 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), and is stimulated indirectly by glucose via the following mechanism:
A glucokinase regulatory protein exists in the nucleus of hepatocytes. 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 5). When glucose levels in the blood (and also in the hepatocyte, as a result of GLUT-2) increase, the glucose causes the release of glucokinase from the regulatory protein, and the enzyme enters the cytosol where it phosphorylates glucose to glucose 6-phosphate. As free glucose levels fall, fructose 6-phosphate causes glucokinase to translocate back into the nucleus and bind to the regulatory protein, thus inhibiting the enzyme s activity.
c. Regulation by insulin: Glucokinase activity in hepatocytes is also increased by insulin. As blood glucose levels rise following a meal, the ? cells of the pancreas are stimulated to release insulin into the portal circulation. Insulin also promotes transcription of the glucokinase gene, resulting in an increase in liver enzyme protein and, therefore, of total glucokinase activity. [Note: The absence of insulin in patients with diabetes causes a deficiency in hepatic glucokinase. This contributes to an inability of the patient to efficiently decrease blood glucose levels.]
B. Isomerization of glucose 6-phosphate
The isomerization of glucose 6-phosphate to fructose 6-phosphateis catalyzed by phosphoglucose isomerase (Figure 6). 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 step of glycolysis (Figure 7). 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 tricarboxylic acid cycle, 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.
2. Regulation by fructose 2,6-bisphosphate: Fructose 2,6-bisphosphate is the most potent activator of PFK-1 (Figure 7). This compound also acts as an inhibitor of fructose 1,6-bisphosphatase, which will discuss later, for the regulation of gluconeogenesis). The reciprocal actions of fructose 2,6-bisphosphate on glycolysis and gluconeogenesis ensure that both pathways are not fully active at the same time. Fructose 2,6-bisphosphate is formed by phosphofructokinase-2 (PFK-2), an enzyme different than phosphofructokinase-1. Fructose 2,6-bisphosphate is converted back to fructose 6-phosphate by fructose bisphosphatase-2 (Figure 8).
[Note: The kinase and phosphatase activities are different domains of one bifunctional polypeptide molecule.]
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 (see Figure 8). 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-bisphos-phate. This results in a decrease in the overall rate of glycolysis and an increase in gluconeogenesis.
D. Cleavage of fructose 1,6-bisphosphate
Aldolase A cleaves fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 7). The reaction is reversible and not regulated. [Note: Aldolase B in the liver and kidney also cleaves fructose 1,6-bisphosphate, and functions in the metabolism of dietary fructose.]
E. Isomerization of dihydroxyacetone phosphate
Triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 7). Dihydroxyacetone phosphate must be isomerized to glyceraldehyde3-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-bisphospho- glycerate by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis (Figure 9). [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, and 2) oxidation of NADH via the respiratory chain.]
1. Synthesis of 1,3-bisphosphoglycerate:
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-bisphosphoglycerate (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. Synthesis of 2,3-bisphosphoglycerate in red blood cells: Some of the 1,3-bisphosphoglycerate is converted to 2,3-bisphospho-glycerate (2,3-BPG) by the action of bisphosphoglycerate mutase (see Figure 9). 2,3-BPG, which is found in only trace amounts in most cells, is present at high concentration in red blood cells. 2,3-BPG is hydrolyzed by a phosphatase to 3-phosphoglycerate, which is also an intermediate in glycolysis (see Figure 9). 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 9). 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. [Note: This is an example of substrate-level phosphorylation, in which the production of a high-energy phosphate is coupled directly to the oxidation of a substrate, instead of resulting from oxidative phosphorylation via the electron transport chain.]
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 (Figure 9).
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 (Figure 9). 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 9). [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 (instead of the more usual feed- back) 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 10).
When blood glucose levels are low, elevated glucagon increases the intracellular level of cAMP, which causes the phosphorylation and inactivation of pyruvate kinase. Therefore, phosphoenolpyruvate 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 bi-concave, 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 the red blood cell result in hemolytic anemia. Among patients exhibiting genetic defects of glycolytic enzymes, about 95 percent show a deficiency in pyruvate kinase, and four percent exhibit phosphoglucose isomerase deficiency. Pyruvate glucose-6-phosphatse dehydrogenase deficiency) of enzymatic-related hemolytic anemia. 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 to 25 percent of normal levels), and on the extent to which the individual s red blood cells compensate by synthesizing increased levels of 2,3-BPG. Almost all individuals with PK deficiency have a mutant enzyme that shows abnormal properties—most: often altered kinetics (Figure 11).
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 12). The formation of lactate is the major fate for pyruvate in red blood cells, lens and cornea of the eye, kidney medulla, testes, and leukocytes.
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 blood stream, 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 (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 end-product. [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 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 provides for the rapid, early detection of oxygen debt in patients. For example, blood lactic acid levels can be used to measure the presence and severity of shock, and to monitor the patient s recovery.
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 (Figure 13). There is no net production or consumption of NADH. Anaerobic glycolysis, although releasing only a small fraction of the energy contained in the glucose molecule, is a valuable source of energy under several conditions, including;
1) when the oxygen supply is limited, as in muscle during intensive exercise; and 2) for tissues with few or no mitochondria, such as the medulla of the kidney, mature erythrocytes, leukocytes, and cells of the lens, cornea, and testes.
2. Aerobic glycolysis: The direct formation and consumption 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.
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 short is, they influence glucose consumption over periods of minutes or hours. Superimposed on these moment-to-moment effects are slower, often more profound, hormonal influences on the amount of enzyme protein synthesized. These effects can result in ten-fold to twenty-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 carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver (Figure 14). 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.
المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .
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