Introduction to metabolism. Investigation of aerobic and anaerobic oxidation of glucose.
Other ways of monosaccharides metabolism.
Carbohydrates have the general molecular formula CH2O, and thus were once thought to represent "hydrated carbon". However, the arrangement of atoms in carbohydrates has little to do with water molecules.
Starch and cellulose are two common carbohydrates. Both are macromolecules with molecular weights in the hundreds of thousands. Both are polymers (hence "polysaccharides"); that is, each is built from repeating units, monomers, much as a chain is built from its links.
The monomers of both starch and cellulose are the same: units of the sugar glucose.
Three common sugars share the same molecular formula: C6H12O6. Because of their six carbon atoms, each is a hexose.
· glucose, "blood sugar", the immediate source of energy for cellular respiration
· galactose, a sugar in milk (and yogurt), and
· fructose, a sugar found in honey.
Although all three share the same molecular formula (C6H12O6), the arrangement of atoms differs in each case. Substances such as these three, which have identical molecular formulas but different structural formulas, are known as structural isomers.
Glucose, galactose, and fructose are "single" sugars or monosaccharides. Two monosaccharides can be linked together to form a "double" sugar or disaccharide.
Three common disaccharides:
· sucrose — common table sugar = glucose + fructose
· lactose — major sugar in milk = glucose + galactose
· maltose — product of starch digestion = glucose + glucose
Although the process of linking the two monomers is rather complex, the end result in each case is the loss of a hydrogen atom (H) from one of the monosaccharides and a hydroxyl group (OH) from the other. The resulting linkage between the sugars is called a glycosidic bond. The molecular formula of each of these disaccharides is
C12H22O11 = 2 C6H12O6 − H2O
All sugars are very soluble in water because of their many hydroxyl groups. Although not as concentrated a fuel as fats, sugars are the most important source of energy for many cells.
Carbohydrates provide the bulk of the calories (4 kcal/gram) in most diets, and starches provide the bulk of that. Starches are polysaccharides.
Starches are polymers of glucose. Two types are found:
· amylose consists of linear, unbranched chains of several hundred glucose residues (units). The glucose residues are linked by a glycosidic bond between their #1 and #4 carbon atoms.
· amylopectin differs from amylose in being highly branched. At approximately every thirtieth residue along the chain, a short side chain is attached by a glycosidic bond to the #6 carbon atom (the carbon above the ring). The total number of glucose residues in a molecule of amylopectin is several thousand.
Starches are insoluble in water and thus can serve as storage depots of glucose. Plants convert excess glucose into starch for storage. The image shows starch grains (lightly stained with iodine) in the cells of the white potato. Rice, wheat, and corn are also major sources of starch in the human diet.
Before starches can enter (or leave) cells, they must be digested. The hydrolysis of starch is done by amylases. With the aid of an amylase (such as pancreatic amylase), water molecules enter at the 1 -> 4 linkages, breaking the chain and eventually producing a mixture of glucose and maltose. A different amylase is needed to break the 1 -> 6 bonds of amylopectin.
Animals store excess glucose by polymerizing it to form glycogen. The structure of glycogen is similar to that of amylopectin, although the branches in glycogen tend to be shorter and more frequent.
Glycogen is broken back down into glucose when energy is needed (a process called glycogenolysis).
· phosphate groups — not water — break the 1 -> 4 linkages
· the phosphate group must then be removed so that glucose can leave the cell.
The liver and skeletal muscle are major depots of glycogen.
There is some evidence that intense exercise and a high-carbohydrate diet ("carbo-loading") can increase the reserves of glycogen in the muscles and thus may help marathoners work their muscles somewhat longer and harder than otherwise. But for most of us, carbo loading leads to increased deposits of fat.
Cellulose is probably the single most abundant organic molecule in the biosphere. It is the major structural material of which plants are made. Wood is largely cellulose while cotton and paper are almost pure cellulose.
Like starch, cellulose is a polysaccharide with glucose as its monomer. However, cellulose differs profoundly from starch in its properties.
· Because of the orientation of the glycosidic bonds linking the glucose residues, the rings of glucose are arranged in a flip-flop manner. This produces a long, straight, rigid molecule.
· There are no side chains in cellulose as there are in starch. The absence of side chains allows these linear molecules to lie close together.
· Because of the many -OH groups, as well as the oxygen atom in the ring, there are many opportunities for hydrogen bonds to form between adjacent chains.
The result is a series of stiff, elongated fibrils — the perfect material for building the cell walls of plants.
This electron micrograph (courtesy of R. D. Preston) shows the cellulose fibrils in the cell wall of a green alga. These long, rigid fibrils are a clear reflection of the nature of the cellulose molecules of which they are composed.
Digestion of Dietary Carbohydrates
Dietary carbohydrate from which humans gain energy enter the body in complex forms, such as disaccharides and the polymers starch (amylose and amylopectin) and glycogen. The polymer cellulose is also consumed but not digested. The first step in the metabolism of digestible carbohydrate is the conversion of the higher polymers to simpler, soluble forms that can be transported across the intestinal wall and delivered to the tissues. The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic pH of 6.8 and contains lingual amylase that begins the digestion of carbohydrates. The action of lingual amylase is limited to the area of the mouth and the esophagus; it is virtually inactivated by the much stronger acid pH of the stomach. Once the food has arrived in the stomach, acid hydrolysis contributes to its degradation; specific gastric proteases and lipases aid this process for proteins and fats, respectively. The mixture of gastric secretions, saliva, and food, known collectively as chyme, moves to the small intestine.
The main polymeric-carbohydrate digesting enzyme of the small intestine is -amylase. This enzyme is secreted by the pancreas and has the same activity as salivary amylase, producing disaccharides and trisaccharides. The latter are converted to monosaccharides by intestinal saccharidases, including maltases that hydrolyze di- and trisaccharides, and the more specific disaccharidases, sucrase, lactase, and trehalase. The net result is the almost complete conversion of digestible carbohydrate to its constituent monosaccharides. The resultant glucose and other simple carbohydrates are transported across the intestinal wall to the hepatic portal vein and then to liver parenchymal cells and other tissues. There they are converted to fatty acids, amino acids, and glycogen, or else oxidized by the various catabolic pathways of cells.
Oxidation of glucose is known as glycolysis.Glucose is oxidized to
either lactate or pyruvate. Under aerobic conditions, the dominant product in
most tissues is pyruvate and the
pathway is known as aerobic glycolysis.
When oxygen is depleted, as for instance during prolonged vigorous exercise,
the dominant glycolytic product in many tissues is lactate and the process is known as anaerobic glycolysis.
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Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the subsequent production of four equivalents of ATP and two equivalents of NADH. Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two moles each of ATP and NADH.
Glucose + 2 ADP + 2 NAD+ + 2 Pi -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+
The NADH generated during glycolysis
is used to fuel mitochondrial ATP synthesis via oxidative
phosphorylation, producing either two or three equivalents of ATP depending
upon whether the glycerol
phosphate shuttle or the malate-aspartate
shuttle is used to transport the electrons from cytoplasmic NADH into the
mitochondria. The net yield from the oxidation of 1 mole of glucose to 2 moles
of pyruvate is, therefore, either 6 or 8 moles of ATP. Complete oxidation of
the 2 moles of pyruvate, through the TCA cycle,
yeilds an additional 30 moles of ATP; the total yield, therefore being either
36 or 38 moles of ATP from the complete oxidation of 1 mole of glucose to CO2
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The pathway of glycolysis can be seen as consisting of 2 separate phases. The first is the chemical priming phase requiring energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, 2 equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is degraded to pyruvate, with the production of 4 equivalents of ATP and 2 equivalents of NADH.
Pathway of glycolysis from glucose to pyruvate. Substrates and products are in blue, enzymes are in green. The two high energy intermediates whose oxidations are coupled to ATP synthesis are shown in red (1,3-bisphosphoglycerate and phosphoenolpyruvate).
The Hexokinase Reaction:
The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P)is the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as hexokinases. The phosphorylation accomplishes two goals: First, the hexokinase reaction converts nonionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars. Second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.
Four mammalian isozymes of hexokinase are known (Types I - IV), with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.
Comparison of the activities of hexokinase and glucokinase. The Km for hexokinase is significantly lower (0.1mM) than that of glucokinase (10mM). This difference ensures that non-hepatic tissues (which contain hexokinase) rapidly and efficiently trap blood glucose within their cells by converting it to glucose-6-phosphate. One major function of the liver is to deliver glucose to the blood and this in ensured by having a glucose phosphorylating enzyme (glucokinase) whose Km for glucose is sufficiently higher that the normal circulating concentration of glucose (5mM).
This feature of hepatic glucokinase allows the liver to buffer blood glucose. After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating glucose. When blood glucose falls to very low levels, tissues such as liver and kidney, which contain glucokinases but are not highly dependent on glucose, do not continue to use the meager glucose supplies that remain available. At the same time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose using their low Km hexokinases, and as a consequence their viability is protected. Under various conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of gluconeogenesis. The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the blood.
The regulation of hexokinase and glucokinase activities is also different. Hexokinases I, II, and III are allosterically inhibited by product (G6P) accumulation, whereas glucokinases are not. The latter further insures liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to peripheral tissues.
The second reaction of glycolysis is an isomerization, in which G6P is converted to fructose 6-phosphate (F6P). The enzyme catalyzing this reaction is phosphohexose isomerase (also known as phosphoglucose isomerase). The reaction is freely reversible at normal cellular concentrations of the two hexose phosphates and thus catalyzes this interconversion during glycolytic carbon flow and during gluconeogenesis.
6-Phosphofructo-1-Kinase (Phosphofructokinase-1, PFK-1):
The next reaction of glycolysis involves the utilization of a second ATP to convert F6P to fructose 1,6-bisphosphate (F1,6BP). This reaction is catalyzed by 6-phosphofructo-1-kinase, better known as phosphofructokinase-1 or PFK-1. This reaction is not readily reversible because of its large positive free energy (G0' = +5.4 kcal/mol) in the reverse direction. Nevertheless, fructose units readily flow in the reverse (gluconeogenic) direction because of the ubiquitous presence of the hydrolytic enzyme, fructose-1,6-bisphosphatase (F-1,6-BPase).
The presence of these two enzymes in the same cell compartment provides an example of a metabolic futile cycle, which if unregulated would rapidly deplete cell energy stores. However, the activity of these two enzymes is so highly regulated that PFK-1 is considered to be the rate-limiting enzyme of glycolysis and F-1,6-BPase is considered to be the rate-limiting enzyme in gluconeogenesis.
Aldolase catalyses the hydrolysis of F1,6BP into two 3-carbon products: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). The aldolase reaction proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis.
Triose Phosphate Isomerase: \
The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase. Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase reaction is pulled in the glycolytic direction by mass action principals.
The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce ATP and NADH. In the first of these reactions, glyceraldehyde-3-P dehydrogenase (G3PDH) catalyzes the NAD+-dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3BPG) and NADH. The G3PDH reaction is reversible, and the same enzyme catalyzes the reverse reaction during gluconeogenesis.
The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase. Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditions. Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-bisphosphoglycerate, 2,3BPG (see Figure below) by the enzyme bisphosphoglycerate mutase. 2,3BPG is an important regulator of hemoglobin's affinity for oxygen. Note that 2,3-bisphosphoglycerate phosphatase degrades 2,3BPG to 3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3BPG shunt thus operates with the expenditure of 1 equivalent of ATP per triose passed through the shunt. The process is not reversible under physiological conditions.
The pathway for 2,3-bisphosphoglycerate (2,3-BPG) synthesis within erythrocytes. Synthesis of 2,3-BPG represents a major reaction pathway for the consumption of glucose in erythrocytes. The synthesis of 2,3-BPG in erythrocytes is critical for controlling hemoglobin affinity for oxygen. Note that when glucose is oxidized by this pathway the erythrocyte loses the ability to gain 2 moles of ATP from glycolytic oxidation of 1,3-BPG to 3-phosphoglycerate via the phosphoglycerate kinase reaction.
Phosphoglycerate Mutase and Enolase:
The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacyl-ester of 3PG to a high-energy form and harvesting the phosphate as ATP. The 3PG is first converted to 2PG by phosphoglycerate mutase and the 2PG conversion to phosphoenoylpyruvate (PEP) is catalyzed by enolase
The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme pyruvate kinase (PK). In this strongly exergonic reaction, the high-energy phosphate of PEP is conserved as ATP. The loss of phosphate by PEP leads to the production of pyruvate in an unstable enol form, which spontaneously tautomerizes to the more stable, keto form of pyruvate. This reaction contributes a large proportion of the free energy of hydrolysis of PEP.
Under aerobic conditions, pyruvate in most cells is further metabolized via the TCA cycle. Under anaerobic conditions and in erythrocytes under aerobic conditions, pyruvate is converted to lactate by the enzyme lactate dehydrogenase (LDH), and the lactate is transported out of the cell into the circulation. The conversion of pyruvate to lactate, under anaerobic conditions, provides the cell with a mechanism for the oxidation of NADH (produced during the G3PDH reaction) to NAD+; which occurs during the LDH catalyzed reaction. This reduction is required since NAD+ is a necessary substrate for G3PDH, without which glycolysis will cease. Normally, during aerobic glycolysis the electrons of cytoplasmic NADH are transferred to mitochondrial carriers of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+.
Aerobic glycolysis generates substantially more ATP per mole of glucose oxidized than does anaerobic glycolysis. The utility of anaerobic glycolysis, to a muscle cell when it needs large amounts of energy, stems from the fact that the rate of ATP production from glycolysis is approximately 100X faster than from oxidative phosphorylation. During exertion muscle cells do not need to energize anabolic reaction pathways. The requirement is to generate the maximum amount of ATP, for muscle contraction, in the shortest time frame. This is why muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis.
The reactions catalyzed by hexokinase, PFK-1 and PK all proceed with a relatively large free energy decrease. These nonequilibrium reactions of glycolysis would be ideal candidates for regulation of the flux through glycolysis. Indeed, in vitro studies have shown all three enzymes to be allosterically controlled.
Regulation of hexokinase, however, is not the major control point in glycolysis. This is due to the fact that large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary. Regulation of PK is important for reversing glycolysis when ATP is high in order to activate gluconeogenesis. As such this enzyme catalyzed reaction is not a major control point in glycolysis. The rate limiting step in glycolysis is the reaction catalyzed by PFK-1.
PFK-1 is a tetrameric enzyme that exist in two conformational states termed R and T that are in equilibrium. ATP is both a substrate and an allosteric inhibitor of PFK-1. Each subunit has two ATP binding sites, a substrate site and an inhibitor site. The substrate site binds ATP equally well when the tetramer is in either conformation. The inhibitor site binds ATP essentially only when the enzyme is in the T state. F6P is the other substrate for PFK-1 and it also binds preferentially to the R state enzyme. At high concentrations of ATP, the inhibitor site becomes occupied and shifting the equilibrium of PFK-1 comformation to that of the T state decreasing PFK-1's ability to bind F6P. The inhibition of PFK-1 by ATP is overcome by AMP which binds to the R state of the enzyme and, therefore, stabilizes the conformation of the enzyme capable of binding F6P. The most important allosteric regulator of both glycolysis and gluconeogenesis is fructose 2,6-bisphosphate, F2,6BP, which is not an intermediate in glycolysis or in gluconeogenesis.
Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase. PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity. (+ve) and (-ve) refer to positive and negative activities, respectively.
The synthesis of F2,6BP is catalyzed by the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase). In the nonphosphorylated form the enzyme is known as PFK-2 and serves to catalyze the synthesis of F2,6BP by phosphorylating fructose 6-phosphate. The result is that the activity of PFK-1 is greatly stimulated and the activity of F-1,6-BPase is greatly inhibited.
Under conditions where PFK-2 is active, fructose flow through the PFK-1/F-1,6-BPase reactions takes place in the glycolytic direction, with a net production of F1,6BP. When the bifunctional enzyme is phosphorylated it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate. The metabolic result of the phosphorylation of the bifunctional enzyme is that allosteric stimulation of PFK-1 ceases, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these two enzymes is gluconeogenic, producing F6P and eventually glucose.
The interconversion of the bifunctional enzyme is catalyzed by cAMP-dependent protein kinase (PKA), which in turn is regulated by circulating peptide hormones. When blood glucose levels drop, pancreatic insulin production falls, glucagon secretion is stimulated, and circulating glucagon is highly increased. Hormones such as glucagon bind to plasma membrane receptors on liver cells, activating membrane-localized adenylate cyclase leading to an increase in the conversion of ATP to cAMP (see diagram below). cAMP binds to the regulatory subunits of PKA, leading to release and activation of the catalytic subunits. PKA phosphorylates numerous enzymes, including the bifunctional PFK-2/F-2,6-BPase. Under these conditions the liver stops consuming glucose and becomes metabolically gluconeogenic, producing glucose to reestablish normoglycemia.
Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the alpha subunit dissociates and binds to and activates adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.
Regulation of glycolysis also occurs at the step catalyzed by pyruvate kinase, (PK). The liver enzyme has been most studied in vitro. This enzyme is inhibited by ATP and acetyl-CoA and is activated by F1,6BP. The inhibition of PK by ATP is similar to the effect of ATP on PFK-1. The binding of ATP to the inhibitor site reduces its affinity for PEP. The liver enzyme is also controlled at the level of synthesis. Increased carbohydrate ingestion induces the synthesis of PK resulting in elevated cellular levels of the enzyme.
A number of PK isozymes have been described. The liver isozyme (L-type), characteristic of a gluconeogenic tissue, is regulated via phosphorylation by PKA, whereas the M-type isozyme found in brain, muscle, and other glucose requiring tissue is unaffected by PKA. As a consequence of these differences, blood glucose levels and associated hormones can regulate the balance of liver gluconeogenesis and glycolysis while muscle metabolism remains unaffected.
In erythrocytes, the fetal PK isozyme has much greater activity than the adult isozyme; as a result, fetal erythrocytes have comparatively low concentrations of glycolytic intermediates. Because of the low steady-state concentration of fetal 1,3BPG, the 2,3BPG shunt (see diagram above) is greatly reduced in fetal cells and little 2,3BPG is formed. Since 2,3BPG is a negative effector of hemoglobin affinity for oxygen, fetal erythrocytes have a higher oxygen affinity than maternal erythrocytes. Therefore, transfer of oxygen from maternal hemoglobin to fetal hemoglobin is favored, assuring the fetal oxygen supply. In the newborn, an erythrocyte isozyme of the M-type with comparatively low PK activity displaces the fetal type, resulting in an accumulation of glycolytic intermediates. The increased 1,3BPG levels activate the 2,3BPG shunt, producing 2,3BPG needed to regulate oxygen binding to hemoglobin.
Genetic diseases of adult erythrocyte PK are known in which the kinase is virtually inactive. The erythrocytes of affected individuals have a greatly reduced capacity to make ATP and thus do not have sufficient ATP to perform activities such as ion pumping and maintaining osmotic balance. These erythrocytes have a short half-life, lyse readily, and are responsible for some cases of hereditary hemolytic anemia.
The liver PK isozyme is regulated by phosphorylation, allosteric effectors, and modulation of gene expression. The major allosteric effectors are F1,6BP, which stimulates PK activity by decreasing its Km(app) for PEP, and for the negative effector, ATP. Expression of the liver PK gene is strongly influenced by the quantity of carbohydrate in the diet, with high-carbohydrate diets inducing up to 10-fold increases in PK concentration as compared to low carbohydrate diets. Liver PK is phosphorylated and inhibited by PKA, and thus it is under hormonal control similar to that described earlier for PFK-2.
Muscle PK (M-type) is not regulated by the same mechanisms as the liver enzyme. Extracellular conditions that lead to the phosphorylation and inhibition of liver PK, such as low blood glucose and high levels of circulating glucagon, do not inhibit the muscle enzyme. The result of this differential regulation is that hormones such as glucagon and epinephrine favor liver gluconeogenesis by inhibiting liver glycolysis, while at the same time, muscle glycolysis can proceed in accord with needs directed by intracellular conditions.
Pyruvate is the branch point molecule of glycolysis. The ultimate fate of pyruvate depends on the oxidation state of the cell. In the reaction catalyzed by G3PDH a molecule of NAD+ is reduced to NADH. In order to maintain the re-dox state of the cell, this NADH must be re-oxidized to NAD+. During aerobic glycolysis this occurs in the mitochondrial electron transport chain generating ATP. Thus, during aerobic glycolysis ATP is generated from oxidation of glucose directly at the PGK and PK reactions as well as indirectly by re-oxidation of NADH in the oxidative phosphorylation pathway. Additional NADH molecules are generated during the complete aerobic oxidation of pyruvate in the TCA cycle. Pyruvate enters the TCA cycle in the form of acetyl-CoA which is the product of the pyruvate dehydrogenase reaction. The fate of pyruvate during anaerobic glycolysis is reduction to lactate.
During anaerobic glycolysis, that period of time when glycolysis is proceeding at a high rate (or in anaerobic organisms), the oxidation of NADH occurs through the reduction of an organic substrate. Erythrocytes and skeletal muscle (under conditions of exertion) derive all of their ATP needs through anaerobic glycolysis. The large quantity of NADH produced is oxidized by reducing pyruvate to lactate. This reaction is carried out by lactate dehydrogenase, (LDH). The lactate produced during anaerobic glycolysis diffuses from the tissues and is transproted to highly aerobic tissues such as cardiac muscle and liver. The lactate is then oxidized to pyruvate in these cells by LDH and the pyruvate is further oxidized in the TCA cycle. If the energy level in these cells is high the carbons of pyruvate will be diverted back to glucose via the gluconeogenesis pathway.
Mammalian cells contain two distinct types of LDH
subunits, termed M and H. Combinations of these different subunits generates
LDH isozymes with different characteristics. The H type subunit predominates in
aerobic tissues such as heart muscle (as the H4 tetramer) while the M subunit
predominates in anaerobic tissues such as skeletal muscle as the M4 tetramer).
H4 LDH has a low Km for pyruvate and also is inhibited by high
levels of pyruvate. The M4 LDH enzyme has a high Km for pyruvate and
is not inhibited by pyruvate. This suggsts that the H-type LDH is utilized for
oxidizing lactate to pyruvate and the M-type the reverse.
Animal cells (primarily hepatocytes) contain the cytosolic enzyme alcohol dehydrogenase (ADH) which oxidizes ethanol to acetaldehyde. Acetaldehyde then enters the mitochondria where it is oxidized to acetate by acetaldehyde dehydrogenase (AcDH).
Acetaldehyde forms adducts with proteins, nucleic acids and other compounds, the results of which are the toxic side effects (the hangover) that are associated with alcohol consumption. The ADH and AcDH catalyzed reactions also leads to the reduction of NAD+ to NADH. The metabolic effects of ethanol intoxication stem from the actions of ADH and AcDH and the resultant cellular imbalance in the NADH/NAD+. The NADH produced in the cytosol by ADH must be reduced back to NAD+ via either the malate-aspartate shuttle or the glycerol-phosphate shuttle. Thus, the ability of an individual to metabolize ethanol is dependent upon the capacity of hepatocytes to carry out eother of these 2 shuttles, which in turn is affected by the rate of the TCA cycle in the mitochondria whose rate of function is being impacted by the NADH produced by the AcDH reaction. The reduction in NAD+ impairs the flux of glucose through glycolysis at the glyceraldehyde-3-phosphate dehydrogenase reaction, thereby limiting energy production. Additionally, there is an increased rate of hepatic lactate production due to the effect of increased NADH on direction of the hepatic lactate dehydrogenase (LDH) reaction. This reverseral of the LDH reaction in hepatocytes diverts pyruvate from gluconeogenesis leading to a reduction in the capacity of the liver to deliver glucose to the blood.
In addition to the negative effects of the altered NADH/NAD+ ratio on hepatic gluconeogenesis, fatty acid oxidation is also reduced as this process requires NAD+ as a cofactor. In fact the opposite is true, fatty acid synthesis is increased and there is an increase in triacylglyceride production by the liver. In the mitocondria, the production of acetate from acetaldehyde leads to increased levels of acetyl-CoA. Since the increased generation of NADH also reduces the activity of the TCA cycle, the acetyl-CoA is diverted to fatty acid synthesis. The reduction in cytosolic NAD+ leads to reduced activity of glycerol-3-phosphate dehydrogenase (in the glcerol 3-phosphate to DHAP direction) resulting in increased levels of glycerol 3-phosphate which is the backbone for the synthesis of the triacylglycerides. Both of these two events lead to fatty acid deposition in the liver leading to fatty liver syndrome.
If for no other reason, it is because of the demands of the brain for oxidizable glucose that the human body exquisitely regulates the level of glucose circulating in the blood. This level is maintained in the range of 5mM.
Nearly all carbohydrates ingested in the diet are converted to glucose following transport to the liver. Catabolism of dietary or cellular proteins generates carbon atoms that can be utilized for glucose synthesis via gluconeogenesis. Additionally, other tissues besides the liver that incompletely oxidize glucose (predominantly skeletal muscle and erythrocytes) provide lactate that can be converted to glucose via gluconeogenesis.
Maintenance of blood glucose homeostasis is of paramount importance to the survival of the human organism. The predominant tissue responding to signals that indicate reduced or elevated blood glucose levels is the liver. Indeed, one of the most important functions of the liver is to produce glucose for the circulation. Both elevated and reduced levels of blood glucose trigger hormonal responses to initiate pathways designed to restore glucose homeostasis. Low blood glucose triggers release of glucagon from pancreatic -cells. High blood glucose triggers release of insulin from pancreatic -cells. Additional signals, ACTH and growth hormone, released from the pituitary act to increase blood glucose by inhibiting uptake by extrahepatic tissues. Glucocorticoids also act to increase blood glucose levels by inhibiting glucose uptake. Cortisol, the major glucocorticoid released from the adrenal cortex, is secreted in response to the increase in circulating ACTH. The adrenal medullary hormone, epinephrine, stimulates production of glucose by activating glycogenolysis in response to stressful stimuli.
Glucagon binding to its' receptors on the surface of liver cells triggers an increase in cAMP production leading to an increased rate of glycogenolysis by activating glycogen phosphorylase via the PKA-mediated cascade. This is the same response hepatocytes have to epinephrine release. The resultant increased levels of G6P in hepatocytes is hydrolyzed to free glucose, by glucose-6-phosphatase, which then diffuses to the blood. The glucose enters extrahepatic cells where it is re-phosphorylated by hexokinase. Since muscle and brain cells lack glucose-6-phosphatase, the glucose-6-phosphate product of hexokinase is retained and oxidized by these tissues.
In opposition to the cellular responses to glucagon (and epinephrine on hepatocytes), insulin stimulates extrahepatic uptake of glucose from the blood and inhibits glycogenolysis in extrahepatic cells and conversely stimulates glycogen synthesis. As the glucose enters hepatocytes it binds to and inhibits glycogen phosphorylase activity. The binding of free glucose stimulates the de-phosphorylation of phosphorylase thereby, inactivating it. Why is it that the glucose that enters hepatocytes is not immediately phosphorylated and oxidized? Liver cells contain an isoform of hexokinase called glucokinase. Glucokinase has a much lower affinity for glucose than does hexokinase. Therefore, it is not fully active at the physiological ranges of blood glucose. Additionally, glucokinase is not inhibited by its product G6P, whereas, hexokinase is inhibited by G6P.
One major response of non-hepatic
tissues to insulin is the recruitment, to the cell surface, of glucose
transporter complexes. Glucose transporters comprise a family of five members, GLUT-1 to GLUT-5. GLUT-1 is
ubiquitously distributed in various tissues. GLUT-2 is found primarily in
intestine, kidney and liver. GLUT-3 is also found in the intestine and GLUT-
Hepatocytes, unlike most other cells,
are freely permeable to glucose and are, therefore, essentially unaffected by
the action of insulin at the level of increased glucose uptake. When blood
glucose levels are low the liver does not compete with other tissues for
glucose since the extrahepatic uptake of glucose is stimulated in response to
insulin. Conversely, when blood glucose levels are high extrahepatic needs are
satisfied and the liver takes up glucose for conversion into glycogen for
future needs. Under conditions of high blood glucose, liver glucose levels will
be high and the activity of glucokinase will be elevated. The G6P produced by
glucokinase is rapidly converted to G1P by phosphoglucomutase, where it can
then be incorporated into glycogen.
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Digestion & Absorption of Proteins & Carbohydrates
Digestion and Absorption of Proteins
1. Humans must ingest proteins, carbohydrates and lipids to maintain tissue and organ functions.
2. Most of these nutrients consist of large polymers that must be broken down before they can be made available to the intestinal cells for transport.
3. Dietary proteins are cleaved by hydrolases with specificity for the peptide bond (peptidases).
4. Endopeptidases (aka Proteases): attack internal protein bonds liberating large peptide fragments.
Exopeptidases: cleave off one amino acid at a time from the....
NH3+, aminopeptidases or COO- terminus, carboxypeptidase.
5. Endo- and Exopeptidases work in concert
The Big Picture:
Protein Digestion and Absorption
Gastric (Stomach) Digestion:
1. Gastric HCl is responsible for the low pH <2 of gastric juice.
2. Gastric acid kills microorganisms and denatures dietary proteins preparing them for hydrolysis by proteases.
3. Gastric juices contain the acid stable proteases of the pepsin family, which produce large peptide fragments and some free amino acids.
4. Protein digestion at this stage is partial, as the amino acids enter the duodenum, they trigger the release of cholectystokinin-pancreozymin (CCK-PZ) into the bloodstream.
This release initiates the secretion of protease zymogens from the pancreas and releases of enteropeptidase in the gut.
1. The pancreatic juice is rich in the proenzymes of endopeptidase and carboxypeptidases.
2. Enteropeptidase activates pancreatic trypsinogen to trypsin.
3. Trypsin autocatalytically activates more trypsinogen and other proenzymes, liberating chymotrypsin, elastase and the carboxypeptidases A and B.
Secretion and Activation of Pancreatic Proteases:
Digestion at the Brush Border (surface of intestinal epithelial cells):
1. Since pancreatic juice does not contain appreciable aminopeptidase activity, final digestion of di- and small peptides depends on brush border enzymes.
2. The surface of intestinal epithelial cells is rich in endopeptidases and aminopeptidases.
3. The end products of cell surface digestion are free amino acids and di- and tripeptides.
1. Following digestion, amino acids and small peptides are co-absorbed w/ sodium via group specific amino acid or peptide transport systems.
2. These processes are carrier mediated, discriminating between natural, L amino acids and D-amino acids, require energy (from the Na+ gradient, Na-K ATPase) and physiologic temperatures.
At least five brush border transport systems exist:
1. neutral amino acids (uncharged aliphatic and aromatic)
2. basic amino acids (
3. acidic amino acids (Asp, Glu)
4. imino acids (Pro), Hydroxyproline)
5. di- and tripeptides
1. Hartnup Disease:
Genetic defect in the neutral amino acid transporter.
Symptoms: dermatitis due to tryptophan malabsorption ("niacin" flush)
Consequences: not serious di- and tripeptide absorption supply minimal amounts of dietarily essential neutral amino acids.
Precursor to kidney stones
Symptoms: painful kidney stone formation due to malabsoprtion of cystine (two disulfide linked cysteines)
Destruction and flattening of the intestinal villi resulting in generalized malabsorption.
Causes: bacterial infection or gluten (contained in certain grains such as wheat and barley) sensitivity.
Digestion and Absorption of Carbohydrates
1. Carbohydrates provide a major component of the daily caloric requirement, ~40%.
2.Distinguish between mono-, di- and polysaccharides.
Monosaccharides- do not need hydrolysis prior to absorption.
Disaccharides- require brush border enzymes.
Polysaccharides- require brush border enzymes, as well as, pancreatic amylase and salivary amylase for digestion.
Hydrolyzed by -amylase into Maltotriose, -Limit Dextrin, Maltose, Glucose
-1,4-glucosidic linkages (non-branching, amylose) and branched chains -1,6 linkages (branch points, amylopectin)
Present in saliva and pancreatic juice.
Specific for internal -1,4-glucosidic bonds.
Brush Border Carbohydrate Digestion:
Final hydrolysis of di- and oligosaccharides to monosaccharides is carried out by -glucosidases on the surface of the small intestine.
Monosaccharides are absorbed by carrier mediated transport.
At least two types are known:
1. Na+monosaccharide transporter
2. Na+ independent, diffusion type monosaccharide transport system
1. Di-, oligo- and polysaccharides that are not hydrolyzed by -amylase and/or brush border enzymes cannot be absorbed.
2. These carbohydrates reach the lower tract of the intestine which contains bacteria.
3. The bacteria utilize many of the remaining carbohydrate, metabolizing them and producing by- products such as: hydrogen gas, methane and carbon dioxide.
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Glycochemistry & Glycobiology
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Carbohydrates are one of three macronutrients that provide the body with energy (protein and fats being the other two). The chemical compounds in carbohydrates are found in both simple and complex forms, and in order for the body to use carbohydrates for energy, food must undergo digestion, absorption, and glycolysis. It is recommended that 55 to 60 percent of caloric intake come from carbohydrates.
Carbohydrates are a main source of energy for the body and are made of carbon, hydrogen, and oxygen. Chlorophyll in plants absorbs light energy from the sun. This energy is used in the process of photosynthesis, which allows green plants to take in carbon dioxide and release oxygen and allows for the production of carbohydrates. This process converts the sun's light energy into a form of chemical energy useful to humans. Plants transform carbon dioxide (CO2) from the air, water (H2O) from the ground, and energy from the sun into oxygen (O2) and carbohydrates (C6H12O6) (6 CO2 + 6 H2O + energy = C6H12O6 + 6 O2). Most carbohydrates have a ratio of 1:2:1 of carbon, hydrogen, and oxygen, respectively.
Humans and other animals obtain carbohydrates by eating foods that contain them. In order to use the energy contained in the carbohydrates, humans must metabolize, or break down, the structure of the molecule in a process that is opposite that of photosynthesis. It starts with the carbohydrate and oxygen and produces carbon dioxide, water, and energy. The body utilizes the energy and water and rids itself of the carbon dioxide.
Simple carbohydrates, or simple sugars, are composed of monosaccharide or disaccharide units. Common monosaccharides (carbohydrates composed of single sugar units) include glucose, fructose, and galactose. Glucose is the most common type of sugar and the primary form of sugar that is stored in the body for energy. It sometimes is referred to as blood sugar or dextrose and is of particular importance to individuals who have diabetes or hypoglycemia. Fructose, the primary sugar found in fruits, also is found in honey and high-fructose corn syrup (in soft drinks) and is a major source of sugar in the diet of Americans. Galactose is less likely than glucose or fructose to be found in nature. Instead, it often combines with glucose to form the disaccharide lactose, often referred to as milk sugar. Both fructose and galactose are metabolized to glucose for use by the body.
Oligosaccharides are carbohydrates made of two to ten monosaccharides. Those composed of two sugars are specifically referred to as disaccharides, or double sugars. They contain two monosaccharides bound by either an alpha bond or a beta bond. Alpha bonds are digestible by the human body, whereas beta bonds are more difficult for the body to break down.
There are three particularly important disaccharides: sucrose, maltose, and lactose. Sucrose is formed when glucose and fructose are held together by an alpha bond. It is found in sugar cane or sugar beets and is refined to make granulated table sugar. Varying the degree of purification alters the
Monosaccharide or disaccharide
Beet sugar (cane sugar)
Disaccharide (fructose and glucose)
Similar to white and powdered sugar, but varied degree of purification
Disaccharide (fructose and glucose)
Similar to white and powdered sugar, but varied degree of purification
High-fructose corn syrup
Very sweet and inexpensive
Fructose and glucose
Disaccharide (glucose and glucose)
Formed by the hydrolysis of starch, but sweeter than starch
Disaccharide (fructose and glucose)
Disaccharide (glucose and galactose)
Made in mammary glands of most lactating animals
Disaccharide (fructose and glucose)
Similar to white and brown sugar, but varied degree of purification
Disaccharide (fructose and glucose)
Similar to brown and powdered sugar, but varied degree of purification
SOURCE: Mahan and Escott-Stump, 2000; Northwestern University; Sizer and Whitney, 1997; and Wardlaw and Kessel, 2002.
final product, but white, brown, and powdered sugars all are forms of sucrose. Maltose, or malt sugar, is composed of two glucose units linked by an alpha bond. It is produced from the chemical decomposition of starch, which occurs during the germination of seeds and the production of alcohol. Lactose is a combination of glucose and galactose. Because it contains a beta bond, it is hard for some individuals to digest in large quantities. Effective digestion requires sufficient amounts of the enzyme lactase.
Complex carbohydrates, or polysaccharides, are composed of simple sugar units in long chains called polymers. Three polysaccharides are of particular importance in human nutrition: starch, glycogen, and dietary fiber.
Starch and glycogen are digestible forms of complex carbohydrates made of strands of glucose units linked by alpha bonds. Starch, often contained in seeds, is the form in which plants store energy, and there are two types: amylose and amylopectin. Starch represents the main type of digestible complex carbohydrate. Humans use an enzyme to break down the bonds linking glucose units, thereby releasing the sugar to be absorbed into the bloodstream. At that point, the body can distribute glucose to areas that need energy, or it can store the glucose in the form of glycogen.
Glycogen is the polysaccharide used to store energy in animals, including humans. Like starch, glycogen is made up of chains of glucose linked by alpha bonds; but glycogen chains are more highly branched than starch. It is this highly branched structure that allows the bonds to be more quickly broken down by enzymes in the body. The primary storage sites for glycogen in the human body are the liver and the muscles.
Another type of complex carbohydrate is dietary fiber. In general, dietary fiber is considered to be polysaccharides that have not been digested at the point of entry into the large intestine. Fiber contains sugars linked by bonds that cannot be broken down by human enzymes, and are therefore
Pastas and whole-grain breads contain complex carbohydrates, which are long strands of glucose molecules. Nutritionists recommend that 55–60 percent of calories come from carbohydrates, and especially complex carbohydrates.
[Photograph by James Noble. Corbis. Reproduced by permission.]
labeled as indigestible. Because of this, most fibers do not provide energy for the body. Fiber is derived from plant sources and contains polysaccharides such as cellulose, hemicellulose, pectin, gums, mucilages, and lignins.
The indigestible fibers cellulose, hemicellulose, and lignin make up the structural part of plants and are classified as insoluble fiber because they usually do not dissolve in water. Cellulose is a nonstarch carbohydrate polymer made of a straight chain of glucose molecules linked by beta bonds and can be found in whole-wheat flour, bran, and vegetables. Hemicellulose is a nonstarch carbohydrate polymer made of glucose, galactose, xylose, and other monosaccharides; it can be found in bran and whole grains. Lignin, a noncarbohydrate polymer containing alcohols and acids, is a woody fiber found in wheat bran and the seeds of fruits and vegetables.
In contrast, pectins, mucilages, and gums are classified as soluble fibers because they dissolve or swell in water. They are not broken down by human enzymes, but instead can be metabolized (or fermented) by bacteria present in the large intestine. Pectin is a fiber made of galacturonic acid and other monosaccharides. Because it absorbs water and forms a gel, it is often used in jams and jellies. Sources of pectin include citrus fruits, apples, strawberries, and carrots. Mucilages and gums are similar in structure. Mucilages are dietary fibers that contain galactose, manose, and other monosaccharides; and gums are dietary fibers that contain galactose, glucuronic acid, and other monosaccharides. Sources of gums include oats, legumes, guar, and barley.
Digestion and Absorption
Carbohydrates must be digested and absorbed in order to transform them into energy that can be used by the body. Food preparation often aids in the digestion process. When starches are heated, they swell and become easier for the body to break down. In the mouth, the enzyme amylase, which is contained in saliva, mixes with food products and breaks some starches into smaller units. However, once the carbohydrates reach the acidic environment of the stomach, the amylase is inactivated. After the carbohydrates have passed through the stomach and into the small intestine, key digestive enzymes are secreted from the pancreas and the small intestine where most digestion and absorption occurs. Pancreatic amylase breaks starch into disaccharides and small polysaccharides, and enzymes from the cells of the small-intestinal wall break any remaining disaccharides into their monosaccharide components. Dietary fiber is not digested by the small intestine; instead, it passes to the colon unchanged.
Sugars such as galactose, glucose, and fructose that are found naturally in foods or are produced by the breakdown of polysaccharides enter into absorptive intestinal cells. After absorption, they are transported to the liver where galactose and fructose are converted to glucose and released into the bloodstream. The glucose may be sent directly to organs that need energy, it may be transformed into glycogen (in a process called glycogenesis) for storage in the liver or muscles, or it may be converted to and stored as fat.
The molecular bonds in food products do not yield high amounts of energy when broken down. Therefore, the energy contained in food is released within cells and stored in the form of adenosine triphosphate (ATP), a high-energy compound created by cellular energy-production systems. Carbohydrates are metabolized and used to produce ATP molecules through a process called glycolysis.
Glycolysis breaks down glucose or glycogen into pyruvic acid through enzymatic reactions within the cytoplasm of the cells. The process results in the formation of three molecules of ATP (two, if the starting product was glucose). Without the presence of oxygen, pyruvic acid is changed to lactic acid, and the energy-production process ends. However, in the presence of oxygen, larger amounts of ATP can be produced. In that situation, pyruvic acid is transformed into a chemical compound called acetyle coenzyme A, a compound that begins a complex series of reactions in the Krebs Cycle and the electron transport system. The end result is a net gain of up to thirty-nine molecules of ATP from one molecule of glycogen (thirty-eight molecules of ATP if glucose was used). Thus, through certain systems, glucose can be used very efficiently in the production of energy for the body.
times, carbohydrates have been incorrectly labeled as "fattening."
Evidence actually supports the consumption of more, rather than less, starchy
foods. Carbohydrates have four calories per gram, while dietary fats
contribute nine per gram, so diets high in complex carbohydrates are likely to
provide fewer calories than diets high in fat. Recommendations are for 55 to 60
percent of total calories to come from carbohydrates (approximately 275 to
diets, such as the Atkins and
is important to consume a minimum amount of carbohydrates to prevent ketosis,
a condition resulting from the breakdown of fat for energy in the absence of carbohydrates.
In this situation, products of fat breakdown, called ketone bodies, build up in
the blood and alter normal pH balance. This can be particularly harmful
to a fetus. To avoid ketosis, daily carbohydrate intake should include a
minimum of 50 to
The exchange system is composed of lists that describe carbohydrate, fat, and protein content, as well as caloric content, for designated portions of specific foods. This system takes into account the presence of more than one type of nutrient in any given food. Exchange lists are especially useful for individuals who require careful diet planning, such as those who monitor intake of calories or certain nutrients. It is particularly useful for diabetics, for whom carbohydrate intake must be carefully controlled, and was originally developed for planning diabetic diets.
Diabetes, Carbohydrate-Modified Diets, and Carbohydrate Counting
Diabetes is a condition that alters the way the body handles carbohydrates. In terms of diet modifications, diabetics can control blood sugar levels by appropriately managing the carbohydrates, proteins, and fats in their meals. The amount of carbohydrates, not necessarily the source, is the primary issue. Blood glucose levels after a meal can be related to the process of food preparation, the amount of food eaten, fat intake, sugar absorption, and the combination of foods in the meal or snack.
One method of monitoring carbohydrate levels—carbohydrate counting—assigns a certain number of carbohydrate grams or exchanges to specific foods. Calculations are used to determine insulin need, resulting in better control of blood glucose levels with a larger variety of foods. Overall, diabetic diets can include moderate amounts of sugar, as long as they are carefully monitored.
Nomenclature of carbohydrates - the naming of carbohydrates - much more information than most of us need
1. Glucose - a monosaccharide - what you measure in your blood.
The chemical formula for glucose is C6H12O6.
2. Sucrose - a disaccharide - table sugar. Is made of one molecule of glucose and one fructose.
3. Starch - amylose and amylopectin - a polysaccharide - potatoes.
Amylose is a straight chain of glucose molecules linked together.
Amylopectin is a branched chain of glucose molecules.
4. Glycolysis is the breakdown of glucose to release its energy in the form of ATP.
Carbohydrate metabolism -
a slide presentation from the
more chemistry than most people will want.