Digestion of Dietary Digestion of Dietary CarbohydratesCarbohydrates

Dietary carbohydrate from which humans gain Dietary carbohydrate from which humans gain energy enter the body in complex forms, such energy enter the body in complex forms, such as disaccharides and the polymers starch as disaccharides and the polymers starch (amylose and amylopectin) and glycogen. The (amylose and amylopectin) and glycogen. The polymer cellulose is also consumed but not polymer cellulose is also consumed but not digested. digested.

The first step in the metabolism of digestible The first step in the metabolism of digestible carbohydrate is the conversion of the higher polymers carbohydrate is the conversion of the higher polymers to simpler, soluble forms that can be transported to simpler, soluble forms that can be transported across the intestinal wall and delivered to the tissues. across the intestinal wall and delivered to the tissues.

The breakdown of polymeric sugars begins in the The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic pH of 6.8 and mouth. Saliva has a slightly acidic pH of 6.8 and contains lingual amylase that begins the digestion of contains lingual amylase that begins the digestion of carbohydrates. The action of lingual amylase is carbohydrates. The action of lingual amylase is limited to the area of the mouth and the esophagus; it limited to the area of the mouth and the esophagus; it is virtually inactivated by the much stronger acid pH of is virtually inactivated by the much stronger acid pH of the stomach. the stomach.

Once the food has arrived in the stomach, acid Once the food has arrived in the stomach, acid hydrolysis contributes to its degradation; specific hydrolysis contributes to its degradation; specific gastric proteases and lipases aid this process for gastric proteases and lipases aid this process for proteins and fats, respectively. The mixture of gastric proteins and fats, respectively. The mixture of gastric secretions, saliva, and food, known collectively as secretions, saliva, and food, known collectively as chyme, moves to the small intestine. chyme, moves to the small intestine.

The main polymeric-carbohydrate digesting enzyme of the The main polymeric-carbohydrate digesting enzyme of the small intestine is small intestine is αα-amylase-amylase. This enzyme is secreted by . This enzyme is secreted by the pancreas and has the same activity as salivary the pancreas and has the same activity as salivary amylase, producing disaccharides and trisaccharides. amylase, producing disaccharides and trisaccharides.

Trisacchariddes are converted to monosaccharides by Trisacchariddes are converted to monosaccharides by intestinal saccharidasesintestinal saccharidases, including , including

maltasesmaltases that hydrolyze di- and trisaccharides, that hydrolyze di- and trisaccharides, and the more specific disaccharidases, and the more specific disaccharidases, sucrasesucrase, , lactaselactase, and , and

trehalasetrehalase. . The net result is the almost complete conversion of digestible The net result is the almost complete conversion of digestible

carbohydrate to its constituent monosaccharides. carbohydrate to its constituent monosaccharides.

The resultant glucose and other simple carbohydrates are The resultant glucose and other simple carbohydrates are transported across the intestinal wall to the hepatic portal transported across the intestinal wall to the hepatic portal vein and then to liver parenchymal cells and other tissues. vein and then to liver parenchymal cells and other tissues. There they are converted to fatty acids, amino acids, and There they are converted to fatty acids, amino acids, and glycogen, or else oxidized by the various catabolic glycogen, or else oxidized by the various catabolic pathways of cells. pathways of cells.

The resultant glucose and other simple The resultant glucose and other simple carbohydrates are transported across the carbohydrates are transported across the intestinal wall to the hepatic portal vein intestinal wall to the hepatic portal vein and then to liver parenchymal cells and and then to liver parenchymal cells and other tissues. There they are converted to other tissues. There they are converted to fatty acids, amino acids, and glycogen, or fatty acids, amino acids, and glycogen, or else oxidized by the various catabolic else oxidized by the various catabolic pathways of cells. pathways of cells.

Glucose cross the plasma membrane of the Glucose cross the plasma membrane of the intestinal cells using a intestinal cells using a NaNa++/glucose/glucose transportertransporter which allows sodium ions and glucose to enter the which allows sodium ions and glucose to enter the cell together (that is, both molecules are passing cell together (that is, both molecules are passing trough the membrane in the same direction: trough the membrane in the same direction: symportersymporter).).

The sodium ions flow down their The sodium ions flow down their concentration gradient while the glucose molecules concentration gradient while the glucose molecules are pumped up theirs. are pumped up theirs.

The sodium ions are pumped back out of the The sodium ions are pumped back out of the cell by the cell by the NaNa++/K/K++ ATPase ATPase in order to maintain their in order to maintain their concentration gradient. concentration gradient.

Glucose is then  released into the Glucose is then  released into the bloodstream by the actionod a bloodstream by the actionod a glucose transporters glucose transporters ( GLUT 5)( GLUT 5) present in the basal membrane of the present in the basal membrane of the endothelial cells . endothelial cells .

Glucose enters most cells by a specific carrier that transports it form the exterior of the cell into the cytosol

Five glucose transporters ( GLUT1,2,3,4,5) are present in mammals, having different kinetic characteristics both all consisting of a single polypeptide with characteristic 12 transmembrane - spanning domains.

Facilitated glucose transport in humans

GLUT1 and GLUT3:

These two types are found in all cells (except

liver and pancreatic beta cells) and are responsible for a basic glucose uptake. The Michaelis-Menten constant Km = 1mM and is slightly lower than the average blood glucose concentration of 4 - 8mM. Thus, glucose slowly, but steadily diffuses into cells where it is metabolized.

GLUT2:

is expressed in liver and beta cells (insulin secretion) of the pancreas. Its Km = 15-20mM is several fold higher than average blood glucose levels of 4 - 8mM glucose. As result glucose entry into liver cells (and beta cells) is normally proportional to the glucose level in blood, while GLUT1/3 systems are saturated and promote a steady glucose influx into cells (e.g. in  neurons).

GLUT4:

is found in adipocytes and skeletal muscle cells and has an affinity of Km = 5mM, right at blood serum levels of glucose (4-8mM). This receptor is upregulated by insulin. High glucose levels, which will saturate the muscle transporter (but not the liver/pancreas type), cause the secretion of insulin. Insulin activates the glut4 gene and more transporter proteins are synthesized and incorporated into the muscle cell membrane, increasing the capacity for glucose transport in this system .

GLUT5GLUT5: : Glucose absorption from small intestine into Glucose absorption from small intestine into mucosal epithelial cells occurs via a Na/glucose mucosal epithelial cells occurs via a Na/glucose symporter. Glucose is actively pumped into the symporter. Glucose is actively pumped into the cells and diffuses via GLUT5 into the portal vein cells and diffuses via GLUT5 into the portal vein blood circulation. Intracellular Nablood circulation. Intracellular Na++ is kept low by is kept low by the Na-K-ATPase on the same basal membrane the Na-K-ATPase on the same basal membrane as GLUT5. as GLUT5. Note that the latter is not a symporter, but rather Note that the latter is not a symporter, but rather a glucose uniporter. These cells not only absorb a glucose uniporter. These cells not only absorb glucose but also Na+ from the gut into the blood glucose but also Na+ from the gut into the blood system. system.

Liver cells store excess glucose as Liver cells store excess glucose as glycogen when blood sugar levels are high glycogen when blood sugar levels are high (just after a high carbohydrate meal) and (just after a high carbohydrate meal) and then breakdown the glycogen as glucose-1-then breakdown the glycogen as glucose-1-phosphate which is converted to glucose-6-phosphate which is converted to glucose-6-phosphate which is finally converted to free phosphate which is finally converted to free glucose. glucose.

The breakdown of glycogen is closely The breakdown of glycogen is closely controlled by hormones. The glucose-1-P controlled by hormones. The glucose-1-P and the glucose-6-P produced by glycogen and the glucose-6-P produced by glycogen breakdown are impermeable to the breakdown are impermeable to the membrane and there is no transport protein membrane and there is no transport protein to allow them to leave the cell. to allow them to leave the cell.

However, there is a transport protein However, there is a transport protein which functions by facilitated diffusion which functions by facilitated diffusion which allows glucose to freely pass back which allows glucose to freely pass back and forth across the membrane.and forth across the membrane.

ATP

High-Energy Cellular CompoundsHigh-Energy Cellular Compounds

ATP ADP + Pi ATP ADP + Pi G = - 12 Kcal

ADP AMP + Pi ADP AMP + Pi G

= - 10 Kcal

AMP AMP AdenosineAdenosine + Pi + Pi G = - 8.2 Kcal

Coenzyme A (CoA)

- MercaptoethylaminePantothenate

ADP

Phosphoenol pyruvate.Phosphoenol pyruvate.

Acetyl phosphate.Acetyl phosphate.

Acetyl – Coenzyme A.Acetyl – Coenzyme A.

A A caloriecalorie (cal) is equivalent to the amount (cal) is equivalent to the amount of of heatheat required to rise the temp of 1 gram required to rise the temp of 1 gram of water from 14.5 to 15.5 ºC.of water from 14.5 to 15.5 ºC.

A A joulejoule (J) is the amount of (J) is the amount of energyenergy needed needed to apply force over a distance of 1 meter.to apply force over a distance of 1 meter.

1 Kcal = 4.184 kJ1 Kcal = 4.184 kJ

NAD

FAD

NAD NADH

FAD FADH2

Oxidative

Phosphorylation

ATP

http://www.med.unibs.it/~marchesi/http://www.med.unibs.it/~marchesi/glycolys.htmlglycolys.html

The Glycolytic PathwayThe Glycolytic Pathway Glycolysis (Greek): Glycolysis (Greek):

GlykosGlykos = sweet = sweet lysislysis = loosening = loosening

Convert glucose to pyruvate.Convert glucose to pyruvate.

Generate 2 mol of ATP.Generate 2 mol of ATP. 10 enzymatic reactions.10 enzymatic reactions.

The enzymes of glycolysis are located in the cytosol.The enzymes of glycolysis are located in the cytosol.

The Energy Derived from The Energy Derived from Glucose OxidationGlucose Oxidation

Aerobic glycolysis of glucose to pyruvate, requires two Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the equivalents of ATP to activate the process, with the subsequent production of four equivalents of ATP and subsequent production of four equivalents of ATP and two equivalents of NADH. two equivalents of NADH.

Thus, conversion of one mole of glucose to two moles of Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two pyruvate is accompanied by the net production of two moles each of ATP and NADH. moles each of ATP and NADH.

Glucose + 2 ADP + 2 NAD+ + 2 Pi --------> Glucose + 2 ADP + 2 NAD+ + 2 Pi --------> 2 Pyruvate + 2 ATP + 2 NADH 2 Pyruvate + 2 ATP + 2 NADH + 2 H++ 2 H+

The NADH generated during glycolysis is used to fuel The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via mitochondrial ATP synthesis via oxidative oxidative phosphorylationphosphorylation, producing either two or three , producing either two or three equivalents of ATP depending upon whether the equivalents of ATP depending upon whether the glycerol phosphate shuttleglycerol phosphate shuttle or the or the malate-aspartatemalate-aspartate shuttle shuttle is used to transport the electrons from is used to transport the electrons from cytoplasmic NADH into the mitochondria. cytoplasmic NADH into the mitochondria.

The net yield from the oxidation of 1 mole of glucose to 2 The net yield from the oxidation of 1 mole of glucose to 2 moles of pyruvate is, therefore, either 6 or 8 moles of moles of pyruvate is, therefore, either 6 or 8 moles of ATP. Complete oxidation of the 2 moles of pyruvate, ATP. Complete oxidation of the 2 moles of pyruvate, through the TCA cycle, yeilds an additional 30 moles of through the TCA cycle, yeilds an additional 30 moles of ATP; the total yield, therefore being either 36 or 38 ATP; the total yield, therefore being either 36 or 38 moles of ATP from the complete oxidation of 1 mole of moles of ATP from the complete oxidation of 1 mole of glucose to COglucose to CO22 and H and H2O. O.

The Individual Reactions of The Individual Reactions of GlycolysisGlycolysis

The pathway of glycolysis can be seen as consisting The pathway of glycolysis can be seen as consisting of 2 separate phases. The first is the of 2 separate phases. The first is the chemical priming chemical priming phasephase requiring energy in the form of ATP, and the requiring energy in the form of ATP, and the second is considered the second is considered the energy-yielding phaseenergy-yielding phase. .

In the first phaseIn the first phase, 2 equivalents of ATP are used to , 2 equivalents of ATP are used to convert glucose to fructose-1,6-bisphosphate (F-1,6-convert glucose to fructose-1,6-bisphosphate (F-1,6-BP). BP).

In the second phaseIn the second phase F-1,6-BP is degraded to F-1,6-BP is degraded to pyruvate, with the production of 4 equivalents of ATP pyruvate, with the production of 4 equivalents of ATP and 2 equivalents of NADH.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). 

GLYCOLYSIS: STEP 1GLYCOLYSIS: STEP 1Glucose to Glucose 6-phosphateGlucose to Glucose 6-phosphate

The ATP-dependent phosphorylation of glucose to form glucose-The ATP-dependent phosphorylation of glucose to form glucose-6-phosphate (G6P) is the first reaction of glycolysis, and is catalyzed by 6-phosphate (G6P) is the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as tissue-specific isoenzymes known as hexokinases. The phosphorylation . The phosphorylation accomplishes two goals: accomplishes two goals: First, the hexokinase reaction converts nonionic glucose into , the hexokinase reaction converts nonionic glucose into an anion that is trapped in the cell, since cells lack transport an anion that is trapped in the cell, since cells lack transport systems systems for phosphorylated sugars. for phosphorylated sugars. Second, the otherwise biologically inert glucose becomes , the otherwise biologically inert glucose becomes activated into a labile form capable of being further activated into a labile form capable of being further metabolized.metabolized.

Four mammalian isozymes of hexokinase are known (Types I - Four mammalian isozymes of hexokinase are known (Types I - IV), with the Type IV isozyme often referred to as IV), with the Type IV isozyme often referred to as glucokinase. . Glucokinase is the form of the enzyme found in hepatocytes. 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.

REACTION MECHANISM OF HEXOKINASE

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 This feature of hepatic glucokinase allows the liver to buffer blood glucose.blood glucose.

After meals, when postprandial blood glucose levels are After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating the liver preferentially to trap and to store circulating glucose. glucose.

When blood glucose falls to very low levels, tissues such as When blood glucose falls to very low levels, tissues such as liver and kidney---which contain glucokinases but are not liver and kidney---which contain glucokinases but are not highly dependent on glucose---do not continue to use the highly dependent on glucose---do not continue to use the meager glucose supplies that remain available. At the same meager glucose supplies that remain available. At the same time, tissues such as the brain, which are critically time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose dependent on glucose, continue to scavenge blood glucose using their low Km hexokinases, and as a consequence using their low Km hexokinases, and as a consequence their viability is protected. their viability is protected.

Under various conditions of glucose deficiency, such as Under various conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of the blood with glucose through the pathway of gluconeogenesis. The levels of glucose produced during . The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the allowing the glucose to pass out of hepatocytes and into the blood.blood.

The regulation of hexokinase and glucokinase activities The regulation of hexokinase and glucokinase activities is also different. Hexokinases I, II, and III are is also different. Hexokinases I, II, and III are allosterically inhibited by product (G6P) accumulation, by product (G6P) accumulation, whereas glucokinases are not. The latter further insures whereas glucokinases are not. The latter further insures liver accumulation of glucose stores during times of liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to utilization when glucose is required to supply energy to peripheral tissues. peripheral tissues.

GLYCOLYSIS: STEP 2GLYCOLYSIS: STEP 2Glucose 6-phosphate to fructose 6-phosphateGlucose 6-phosphate to fructose 6-phosphate

G6P is converted to fructose-6-phosphate (F6P). The enzyme G6P is converted to fructose-6-phosphate (F6P). The enzyme catalyzing this reaction is catalyzing this reaction is phosphohexose isomerase (also known as phosphoglucose isomerase). The reaction (an aldose to ketose isomerisation, is freely reversible The reaction (an aldose to ketose isomerisation, is freely reversible at normal cellular concentrations of the two hexose phosphates and at normal cellular concentrations of the two hexose phosphates and thus catalyzes this interconversion during glycolytic carbon flow and thus catalyzes this interconversion during glycolytic carbon flow and during gluconeogenesis. during gluconeogenesis.

  Step 3:

6-Phosphofructo-1-Kinase (Phosphofructokinase-1, PFK-1)(Phosphofructokinase-1, PFK-1)

The next reaction of glycolysis involves the utilization of a second The next reaction of glycolysis involves the utilization of a second ATP to convert F6P to fructose-1,6-bisphosphate (F-1,6-BP). This ATP to convert F6P to fructose-1,6-bisphosphate (F-1,6-BP). This reaction is catalyzed by reaction is catalyzed by 6-phosphofructo-1-kinase, better known as , better known as phosphofructokinase-1 or PFK-1. .

This reaction This reaction is not readily reversibleis not readily reversible because of its large because of its large positive free energy (positive free energy (GG00' = +5.4 kcal/mol) in the reverse ' = +5.4 kcal/mol) in the reverse direction. Nevertheless, fructose units readily flow in the direction. Nevertheless, fructose units readily flow in the reverse (gluconeogenic) direction because of the reverse (gluconeogenic) direction because of the ubiquitous presence of the hydrolytic enzyme, ubiquitous presence of the hydrolytic enzyme, fructose-fructose-1,6-bisphosphatase1,6-bisphosphatase (F-1,6-BPase) (F-1,6-BPase)..

The presence of these two enzymes in the same cell The presence of these two enzymes in the same cell compartment provides an example of a compartment provides an example of a metabolic futile cycle, which if unregulated would rapidly deplete cell , which if unregulated would rapidly deplete cell energy stores. However, the activity of these two enzymes energy stores. However, the activity of these two enzymes is so highly regulated that PFK-1 is considered to be the is so highly regulated that PFK-1 is considered to be the rate-limiting enzyme of glycolysis and F-1,6-BPase is rate-limiting enzyme of glycolysis and F-1,6-BPase is considered to be the rate-limiting enzyme in considered to be the rate-limiting enzyme in gluconeogenesis. gluconeogenesis.

Step 4: AldolaseStep 4: Aldolase

Aldolase catalyses the hydrolysis of F-1,6-BP into two 3-Aldolase catalyses the hydrolysis of F-1,6-BP into two 3-carbon products: dihydroxyacetone phosphate (DHAP) and carbon products: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase reaction glyceraldehyde-3-phosphate (G3P). The aldolase reaction proceeds readily in the reverse direction, being utilized for proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis.both glycolysis and gluconeogenesis.

Voet 16-9

Step 5:Step 5:Triose Phosphate Isomerase

The two products of the aldolase reaction equilibrate The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by readily in a reaction catalyzed by triose phosphate triose phosphate isomeraseisomerase. Succeeding reactions of glycolysis utilize . Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase reaction is pulled G3P as a substrate; thus, the aldolase reaction is pulled in the glycolytic direction by mass action principals. in the glycolytic direction by mass action principals.

Step 6:Step 6: Glyceraldehyde -3-Phosphate Glyceraldehyde -3-Phosphate

DehydrogenaseDehydrogenase

The second phase of glucose catabolism features the The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce ATP and energy-yielding glycolytic reactions that produce ATP and NADH. NADH.

In the first of these reactions, In the first of these reactions, glyceraldehyde-3-P glyceraldehyde-3-P dehydrogenasedehydrogenase (G3PDH) (G3PDH) catalyzes the NAD catalyzes the NAD++-dependent -dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3-BPG) oxidation of G3P to 1,3-bisphosphoglycerate (1,3-BPG) and NADH. The G3PDH reaction is reversible, and the and NADH. The G3PDH reaction is reversible, and the same enzyme catalyzes the reverse reaction during same enzyme catalyzes the reverse reaction during gluconeogenesis. gluconeogenesis.

NAD+ +

NADH + H+

Pi

Glyceraldehyde3-phosphate dehydrogenase

A high-energy phosphate compound is generated In this oxidation – reduction reaction.

The enzymatic mechanism ofGlyceraldehyde -3-phosphate dehydrogenase.

(Voet 16-14)

Step 7: Step 7: Phosphoglycerate KinasePhosphoglycerate Kinase

The high-energy phosphate of 1,3-BPG is used to form ATP and 3-The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate (3PG) by the enzyme phosphoglycerate kinasephosphoglycerate kinase. .

Note that this is the only reaction of glycolysis or gluconeogenesis Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditions. that involves ATP and yet is reversible under normal cell conditions. Associated with the phosphoglycerate kinase pathway is an Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-BPG by the important reaction of erythrocytes, the formation of 2,3-BPG by the enzyme enzyme bisphosphoglycerate mutasebisphosphoglycerate mutase. 2,3-BPG is an important . 2,3-BPG is an important regulator of hemoglobin's affinity for oxygen. Note that regulator of hemoglobin's affinity for oxygen. Note that 2,3-2,3-bisphosphoglycerate phosphatasebisphosphoglycerate phosphatase degrades 2,3-BPG to 3- degrades 2,3-BPG to 3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3-BPG phosphoglycerate, a normal intermediate of glycolysis. The 2,3-BPG shunt thus operates with the expenditure of 1 equivalent of ATP per shunt thus operates with the expenditure of 1 equivalent of ATP per triose passed through the shunt. The process is not reversible under triose passed through the shunt. The process is not reversible under physiological conditions. physiological conditions.

Step 8: Step 8: Phosphoglycerate Mutase Phosphoglycerate Mutase andand

Step 9: EnolaseStep 9: Enolase

The remaining reactions of glycolysis are aimed at The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacyl-converting the relatively low energy phosphoacyl-ester of 3-PG to a high-energy form and harvesting ester of 3-PG to a high-energy form and harvesting the phosphate as ATP. the phosphate as ATP.

The 3-PG is first converted to 2-PG by The 3-PG is first converted to 2-PG by phosphoglycerate mutasephosphoglycerate mutase and the 2-PG conversion and the 2-PG conversion to phosphoenoylpyruvate (PEP) is catalyzed by to phosphoenoylpyruvate (PEP) is catalyzed by enolase.enolase.

PHOSPHOGLYCEROMUTASE

ENOLASE

Step 10: Pyruvate Kinase

The final reaction of aerobic The final reaction of aerobic glycolysis is catalyzed by glycolysis is catalyzed by the highly regulated enzyme the highly regulated enzyme pyruvate kinase (PK)). In . In this strongly exergonic this strongly exergonic reaction, the high- energy reaction, the high- energy phosphate of PEP is phosphate of PEP is conserved as ATP. The loss conserved as ATP. The loss of phosphate by PEP leads of phosphate by PEP leads to the production of pyruvate to the production of pyruvate in an unstable enol form, in an unstable enol form, which spontaneously which spontaneously tautomerizes to the more tautomerizes to the more stable, keto form of stable, keto form of pyruvate. pyruvate. This reaction contributes a large proportion of the free energy of hydrolysis of PEP.

PYRUVATE KINASE

The high-energy phosphate of 1,3-BPG is used to form ATP and 3-The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate (3PG) by the enzyme phosphoglycerate kinasephosphoglycerate kinase. .

Note that this is the only reaction of glycolysis or gluconeogenesis Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditionsthat involves ATP and yet is reversible under normal cell conditions. . Associated with the phosphoglycerate kinase pathway is an Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-BPG by the important reaction of erythrocytes, the formation of 2,3-BPG by the enzyme enzyme bisphosphoglycerate mutasebisphosphoglycerate mutase. . 2,3-BPG is an important 2,3-BPG is an important regulator of hemoglobin's affinity for oxygen.regulator of hemoglobin's affinity for oxygen. Note that Note that 2,3-2,3-bisphosphoglycerate phosphatasebisphosphoglycerate phosphatase degrades 2,3-BPG to 3- degrades 2,3-BPG to 3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3-BPG phosphoglycerate, a normal intermediate of glycolysis. The 2,3-BPG shunt thus operates with the expenditure of 1 equivalent of ATP per shunt thus operates with the expenditure of 1 equivalent of ATP per triose passed through the shunt. The process is not reversible under triose passed through the shunt. The process is not reversible under physiological conditions. physiological conditions.

The 2,3-Bisphosphoglycerate The 2,3-Bisphosphoglycerate Pathway in Erythrocytes Pathway in Erythrocytes

Synthesis of 2,3-BPG represents a major reaction pathway Synthesis of 2,3-BPG represents a major reaction pathway for the consumption of glucose in erythrocytes.for the consumption of glucose in erythrocytes.

The synthesis of 2,3-BPG in erythrocytes is critical for The synthesis of 2,3-BPG in erythrocytes is critical for controlling hemoglobin affinity for oxygen. controlling hemoglobin affinity for oxygen.

Note that when glucose is oxidized by this pathway the Note that when glucose is oxidized by this pathway the erythrocyte loses the ability to gain 2 moles of ATP from erythrocyte loses the ability to gain 2 moles of ATP from glycolytic oxidation of 1,3-BPG to 3-phosphoglycerate via glycolytic oxidation of 1,3-BPG to 3-phosphoglycerate via the the phosphoglycerate kinasephosphoglycerate kinase reaction.  reaction. 

The pathway for 2,3-bisphosphoglycerate The pathway for 2,3-bisphosphoglycerate ((2,3-BPG2,3-BPG) synthesis within erythrocytes.) synthesis within erythrocytes.

The compound 2,3-bisphosphoglycerate (2,3-BPG), derived from the The compound 2,3-bisphosphoglycerate (2,3-BPG), derived from the glycolytic intermediate 1,3-bisphosphoglycerate, is a potent allosteric glycolytic intermediate 1,3-bisphosphoglycerate, is a potent allosteric effector on the oxygen binding properties of hemoglobin. effector on the oxygen binding properties of hemoglobin.

In the deoxygenated In the deoxygenated TT conformer, a cavity capable of binding 2,3-BPG conformer, a cavity capable of binding 2,3-BPG forms in the center of the molecule. 2,3-BPG can occupy this cavity forms in the center of the molecule. 2,3-BPG can occupy this cavity stabilizing the T statestabilizing the T state. Conversely, when 2,3-BPG is not available, or not . Conversely, when 2,3-BPG is not available, or not bound in the central cavity, Hb can be converted to HbObound in the central cavity, Hb can be converted to HbO22 more readily. more readily. Thus, like increased hydrogen ion concentration, Thus, like increased hydrogen ion concentration, increased 2,3-BPG increased 2,3-BPG concentration favors conversion of R form Hb to T form Hb and decreases concentration favors conversion of R form Hb to T form Hb and decreases the amount of oxygen bound by Hb at any oxygen concentrationthe amount of oxygen bound by Hb at any oxygen concentration. .

Hemoglobin molecules differing in subunit composition are known to have Hemoglobin molecules differing in subunit composition are known to have different 2,3-BPG binding properties with correspondingly different different 2,3-BPG binding properties with correspondingly different allosteric responses to 2,3-BPG. For example, allosteric responses to 2,3-BPG. For example, HbF (the fetal form of hemoglobin) binds 2,3-BPG much less avidly than HbA (the adult form of binds 2,3-BPG much less avidly than HbA (the adult form of hemoglobin) with the result that HbF in fetuses of pregnant women binds hemoglobin) with the result that HbF in fetuses of pregnant women binds oxygen with greater affinity than the mothers HbA, thus giving the fetus oxygen with greater affinity than the mothers HbA, thus giving the fetus preferential access to oxygen carried by the mothers circulatory system.preferential access to oxygen carried by the mothers circulatory system.

Homolactic fermentation (in muscle).

Alcoholic fermentation (in bacteria).

Maintaining Redox Balance:Maintaining Redox Balance:The Diverse Fates of PyruvateThe Diverse Fates of Pyruvate

The The CoriCori Cycle Cycle

The Entry of The Entry of FructoseFructose and and GalactoseGalactose into into GlycolysisGlycolysis

Excessive Fructose Depletes Excessive Fructose Depletes Liver PLiver Pi .i .

Fructose Intolerance.Fructose Intolerance. Results from a deficiency of Results from a deficiency of

Type B aldolase.Type B aldolase.

There are no catabolic pathways to

metabolize galactose,

so the strategy is to convert galactose

into a metabolite of glucose.

MannoseMannose________________________________________________________

Glycogen MetabolismGlycogen Metabolism

The structure of glycogen. (a) Molecular formula. In the actual molecule the chains are much longer than shown. (b) Schematic diagram illustrating its branched structure. Branch points in the actual molecule are separated by 8 to 12 glucosyl units. Note that the molecule, no matter how big, has but one reducing end.

Contain up to 120,000 glucose units.

Muscle contain 1 - 2 % glycogen by weight.

Liver contain 10 % glycogen by weight (12 h energy supply for the body).

A photomicrograph showing the glycogen granules in the cytoplasm of liver cell. The glycogen content of liver may reach as high as 10% of its net weight.

glycogen granules

(Also contain the enzymes that catalyze glycogen synthesis and degradation as well as some of the enzymes that regulate these process.

Fat globule

Mitchondrion

Why does the body go to such metabolic effort to use glycogen for energy storage when fat, which is far more abundant in the body, seemingly serves the same purpose?

The answer:

1- Muscle cannot mobilize fat as rapidly as they can glycogen.

2- The fatty acids residues of fat cannot be metabolized anaerobically.

3- Animals cannot convert fatty acids to glucose, so fat metabolism alone cannot adequately maintain essential blood glucose levels.

Glycogen BreakdownGlycogen Breakdown

Requires Three EnzymesRequires Three Enzymes1- Glycogen Phosphorylase 1- Glycogen Phosphorylase

(or simply phosphorylase)(or simply phosphorylase)

Glycogen + Pi ↔ glycogen + glucose -1- phosphate (n) ( n-1 )

Only release a glucose unit that is at least five units from a branch point.

Pyridoxal phosphate is an essential cofactor for phosphorylase.

Phosphorylase contains pyridoxal -5- phosphate (PLP)

2- Glycogen Debranching Enzyme. Removes glycogen’s branches, thereby permitting the glycogen phosphorylase reaction to go to completion.

It also hydrolyzes α (1 → 6 ) – linked glucosyl units to yield glucose.

Consequently, 90% of glycogen’s glucose residues are converted to G1P. The remaining 10%, those at the branch point, are converted to glucose.

Thus debranching enzyme has active sites for both the transferase reaction and the α (1 → 6 ) –glucosidase reaction.

The presence of two independent catalytic activities on the same enzyme improves the efficiency of the debranching process.

3- Phosphoglucomutase.

GLYCOGEN

PhosphorylaseDebranching Enzyme

G1PGlucose

Phosphoglucomutase

G6P

Phosphoglucomutase.

glycogen

Glycogen n-1

Glucose 1- P

Glucose 6 - P

Glucose

Pyruvate

Lactate CO2 + H2O

Ribose + NADPH

Phosphorylase

Phosphoglucomutase

Glucose 6-phosphatase

LiverMuscle, Brain

Pentose PhosphatePathway

Glycogen SynthesisGlycogen Synthesis

Synthesis of glycogen from glucose is carried out the enzyme Synthesis of glycogen from glucose is carried out the enzyme glycogen synthase.

This enzyme utilizes UDP-glucose as one substrate and the non-This enzyme utilizes UDP-glucose as one substrate and the non-reducing end of glycogen as another. reducing end of glycogen as another.

The activation of glucose to be used for glycogen synthesis is carried The activation of glucose to be used for glycogen synthesis is carried out by the enzyme out by the enzyme UDP-glucose pyrophosphorylase. This enzyme . This enzyme exchanges the phosphate on C-1 of glucose-1-phosphate for UDP. exchanges the phosphate on C-1 of glucose-1-phosphate for UDP.

The energy of the phospho-glycosyl bond of UDP-glucose is utilized by The energy of the phospho-glycosyl bond of UDP-glucose is utilized by glycogen synthase to catalyze the incorporation of glucose into glycogen synthase to catalyze the incorporation of glucose into glycogen. UDP is subsequently released from the enzyme. glycogen. UDP is subsequently released from the enzyme.

The The -1,6 branches in glucose are produced by -1,6 branches in glucose are produced by amylo-(1,4 - 1,6)-transglycosylase, also termed the , also termed the branching enzymebranching enzyme. This enzyme . This enzyme transfers a terminal fragment of 6-7 glucose residues (from a polymer transfers a terminal fragment of 6-7 glucose residues (from a polymer at least 11 glucose residues long) to an internal glucose residue at the at least 11 glucose residues long) to an internal glucose residue at the C-6 hydroxyl position. C-6 hydroxyl position.

Until recently, the source of the first glycogen molecule that might act Until recently, the source of the first glycogen molecule that might act as a primer in glycogen synthesis was unknown. Recently it has been as a primer in glycogen synthesis was unknown. Recently it has been discovered that a protein known as discovered that a protein known as glycogenin is located at the core glycogenin is located at the core of glycogen molecules. Glycogenin has the unusual property of of glycogen molecules. Glycogenin has the unusual property of catalyzing its own glycosylation, attaching C-1 of a UDP-glucose catalyzing its own glycosylation, attaching C-1 of a UDP-glucose to a tyrosine residue on the enzyme. The attached glucose is to a tyrosine residue on the enzyme. The attached glucose is believed to serve as the primer required by glycogen synthase. believed to serve as the primer required by glycogen synthase.

Three Enzymes are involveThree Enzymes are involve

1- UDP-Glucose 1- UDP-Glucose Pyrophosphorylase.Pyrophosphorylase.

2- Glycogen Synthase.2- Glycogen Synthase.

3- Glycogen Branching.3- Glycogen Branching.

Glycogen Storage DiseasesGlycogen Storage Diseases

Since glycogen molecules can become enormously large, an inability to degrade Since glycogen molecules can become enormously large, an inability to degrade glycogen can cause cells to become pathologically engorged; it can also lead to the glycogen can cause cells to become pathologically engorged; it can also lead to the functional loss of glycogen as a source of cell energy and as a blood glucose buffer. functional loss of glycogen as a source of cell energy and as a blood glucose buffer. Although glycogen storage diseases are quite rare, their effects can be most Although glycogen storage diseases are quite rare, their effects can be most dramatic. The debilitating effect of many glycogen storage diseases depends on the dramatic. The debilitating effect of many glycogen storage diseases depends on the severity of the mutation causing the deficiency. In addition, although the glycogen severity of the mutation causing the deficiency. In addition, although the glycogen storage diseases are attributed to specific enzyme deficiencies, other events can storage diseases are attributed to specific enzyme deficiencies, other events can cause the same characteristic symptoms. For example, Type I glycogen storage cause the same characteristic symptoms. For example, Type I glycogen storage disease (disease (von Gierke's disease) is attributed to lack of glucose-6-phosphatase. ) is attributed to lack of glucose-6-phosphatase. However, this enzyme is localized on the cisternal surface of the endoplasmic However, this enzyme is localized on the cisternal surface of the endoplasmic reticulum (ER); in order to gain access to the phosphatase, glucose-6-phosphate reticulum (ER); in order to gain access to the phosphatase, glucose-6-phosphate must pass through a specific translocase in the ER membrane. Mutation of either the must pass through a specific translocase in the ER membrane. Mutation of either the phosphatase or the translocase makes transfer of liver glycogen to the blood a very phosphatase or the translocase makes transfer of liver glycogen to the blood a very limited process. Thus, mutation of either gene leads to symptoms associated with von limited process. Thus, mutation of either gene leads to symptoms associated with von Gierke's disease, which occurs at a rate of about 1 in 200,000 people. Gierke's disease, which occurs at a rate of about 1 in 200,000 people.

Several glycogenoses are the result of deficiencies in enzymes of glycolysis whose Several glycogenoses are the result of deficiencies in enzymes of glycolysis whose symptoms and signs are similar to those seen in type V glycogen storage disease. symptoms and signs are similar to those seen in type V glycogen storage disease. These include deficiencies in muscle phosphglycerate kinase and muscle pyruvate These include deficiencies in muscle phosphglycerate kinase and muscle pyruvate kinase as well as deficiencies in fructose 1,6-bisphosphatase, lactate dehydrogenase kinase as well as deficiencies in fructose 1,6-bisphosphatase, lactate dehydrogenase and phosphoglycerate mutase. and phosphoglycerate mutase.

GluconeogenesisGluconeogenesis

Gluconeogenesis is the biosynthesis of new glucose, Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen). (i.e. not glucose from glycogen).

The production of glucose from other metabolites is The production of glucose from other metabolites is necessary for use as a fuel source by the necessary for use as a fuel source by the brain, brain, testes, erythrocytestestes, erythrocytes and and kidney medulla since kidney medulla since glucose is the sole energy source for these glucose is the sole energy source for these organs. organs.

During starvation, however, the brain can derive During starvation, however, the brain can derive energy from ketone bodies which are converted energy from ketone bodies which are converted to acetyl-CoA. to acetyl-CoA.

Synthesis of glucose from three and four carbon Synthesis of glucose from three and four carbon precursors is essentially a reversal of glycolysis. precursors is essentially a reversal of glycolysis. The relevant features of the pathway of The relevant features of the pathway of gluconeogenesis are diagrammed below. gluconeogenesis are diagrammed below.

Figure 16.24. Pathway of Gluconeogenesis. The distinctive reactions and enzymes of this pathway are shown in red. The other reactions are common to glycolysis. The enzymes for gluconeogenesis are located in the cytosol, except for pyruvate carboxylase (in the mitochondria) and glucose 6-phosphatase (membrane bound in the endoplasmic reticulum). The entry points for lactate, glycerol, and amino acids are shown.

Gluconeogenesis Is Not a Reversal of GlycolysisGluconeogenesis Is Not a Reversal of GlycolysisIn glycolysis, glucose is converted into pyruvate; in gluconeogenesis, In glycolysis, glucose is converted into pyruvate; in gluconeogenesis,

pyruvate is converted into glucose. However, pyruvate is converted into glucose. However, gluconeogenesis is not a gluconeogenesis is not a reversal of glycolysisreversal of glycolysis. Several reactions must differ because the equilibrium . Several reactions must differ because the equilibrium of glycolysis lies far on the side of pyruvate formation. The actual of glycolysis lies far on the side of pyruvate formation. The actual GG for the for the

formation of pyruvate from glucose is about -20 kcal molformation of pyruvate from glucose is about -20 kcal mol -1-1 (-84 kJ mol (-84 kJ mol-1-1) under ) under typical cellular conditions. Most of the decrease in free energy in glycolysis typical cellular conditions. Most of the decrease in free energy in glycolysis

takes place in the three essentially irreversible steps catalyzed by takes place in the three essentially irreversible steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.hexokinase, phosphofructokinase, and pyruvate kinase.

In gluconeogenesis, the following new steps bypass these virtually In gluconeogenesis, the following new steps bypass these virtually irreversible reactions of glycolysis:irreversible reactions of glycolysis:

1. 1. Phosphoenolpyruvate is formed from pyruvate by way Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetateof oxaloacetate through the action of pyruvate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase carboxylase and phosphoenolpyruvate carboxykinase

2. 2. Fructose 6-phosphate is formed from Fructose 6-phosphate is formed from fructose 1,6-bisphosphate by hydrolysis of fructose 1,6-bisphosphate by hydrolysis of the phosphate ester at carbon 1the phosphate ester at carbon 1. Fructose . Fructose 1,6-bisphosphatase catalyzes this 1,6-bisphosphatase catalyzes this exergonic hydrolysis. exergonic hydrolysis.

Fructose1,6-bisphosphatase

3. 3. Glucose is formed by hydrolysis of Glucose is formed by hydrolysis of glucose 6-phosphateglucose 6-phosphate in a reaction in a reaction catalyzed by glucose 6-phosphatase. catalyzed by glucose 6-phosphatase.

The three reactions of glycolysis that proceed with a The three reactions of glycolysis that proceed with a large negative free energy change are bypassed during large negative free energy change are bypassed during gluconeogenesis by using different enzymesgluconeogenesis by using different enzymes. . These These three are: three are: the pyruvate kinase, the pyruvate kinase, phosphofructokinase-1phosphofructokinase-1((PFK-1PFK-1) ) and hexokinaseand hexokinase//glucokinase catalyzed reactionsglucokinase catalyzed reactions. .

In the liver or kidney cortex and in some cases skeletal In the liver or kidney cortex and in some cases skeletal muscle, the glucose-6-phosphate muscle, the glucose-6-phosphate ((G6PG6P) ) produced by produced by gluconeogenesis can be incorporated into glycogengluconeogenesis can be incorporated into glycogen. . In In this case the third bypass occurs at the glycogen this case the third bypass occurs at the glycogen phosphorylase catalyzed reactionphosphorylase catalyzed reaction. . Since skeletal muscle Since skeletal muscle lacks glucose-6-phosphatase it cannot deliver free lacks glucose-6-phosphatase it cannot deliver free glucose to the blood and undergoes gluconeogenesis glucose to the blood and undergoes gluconeogenesis exclusively as a mechanism to generate glucose for exclusively as a mechanism to generate glucose for storage as glycogenstorage as glycogen. .

Pyruvate to Phosphoenolpyruvate Pyruvate to Phosphoenolpyruvate ((PEPPEP)), , Bypass 1Bypass 1

Conversion of pyruvate to PEP requires the action of Conversion of pyruvate to PEP requires the action of two mitochondrial enzymestwo mitochondrial enzymes. .

The first is an ATPThe first is an ATP--requiring reaction catalyzed by pyruvate requiring reaction catalyzed by pyruvate carboxylase, (PC). As the name of the enzyme implies, carboxylase, (PC). As the name of the enzyme implies, pyruvate is carboxylated to form oxaloacetate (OAA). pyruvate is carboxylated to form oxaloacetate (OAA). The CO2 in this reaction is in the form of bicarbonate The CO2 in this reaction is in the form of bicarbonate (HCO3-) . This reaction is an anaplerotic reaction since it (HCO3-) . This reaction is an anaplerotic reaction since it can be used to fill-up the TCA cycle. can be used to fill-up the TCA cycle.

The second enzyme in the conversion of pyruvate to The second enzyme in the conversion of pyruvate to PEP is PEP carboxykinase (PEPCK). PEPCK requires PEP is PEP carboxykinase (PEPCK). PEPCK requires GTP in the decarboxylation of OAA to yield PEP. Since GTP in the decarboxylation of OAA to yield PEP. Since PC incorporated CO2 into pyruvate and it is PC incorporated CO2 into pyruvate and it is subsequently released in the PEPCK reaction, no net subsequently released in the PEPCK reaction, no net fixation of carbon occurs. Human cells contain almost fixation of carbon occurs. Human cells contain almost equal amounts of mitochondrial and cytosolic PEPCK so equal amounts of mitochondrial and cytosolic PEPCK so this second reaction can occur in either cellular this second reaction can occur in either cellular compartment. compartment.