1. Carbohydrate metabolism and Glycolysis University of Babylon College of pharmacy 3 rd class – second semester Biochemistry - carbohydrate metabolism Dr. Abdulhussien M. K. Aljebory 13/15/20179/1/20171

2. GLYCOLYSIS There are two types of metabolism : 1-Aerobic respiration is relate to the structure of a mitochondrion. Glycolysis and Krebs cycle synthesis 10% of ATP. Electron transport chain synthesis 90% of ATP. 2- un aerobic respiration - formation of lactate. Respiration involves three stages: 1. Glycolysis harvests chemical energy by oxidizing glucose to pyruvate and produces about 5% of ATP () 1. Glycolysis harvests chemical energy by oxidizing glucose to pyruvate and produces about 5% of ATP (in cytoplasm) 2. Krebs cycle completes the energy-yielding oxidation of organic molecules and produces about 5% of ATP() 2. Krebs cycle completes the energy-yielding oxidation of organic molecules and produces about 5% of ATP (in mitochondrial matrix) 3. Electron transport chain to synthesisATP and produces about 90% of ATP( 3. Electron transport chain to synthesis ATP and produces about 90% of ATP (inner mitochondrial membrane).  Cellular respiration generates many ATP molecules. For each sugar molecule it oxidizes ( 38 ATP molecule ). 9/1/20172

3. Respiration involves glycolysis, the Krebs cycle, and electron transport Respiration occurs in three metabolic stages: glycolysis, the Krebs cycle, and the electron transport chain and oxidative phosphorylation. Respiration occurs in three metabolic stages: glycolysis, the Krebs cycle, and the electron transport chain and oxidative phosphorylation. Glycolysis occurs in the cytoplasm. Glycolysis occurs in the cytoplasm. –It begins catabolism by breaking glucose into two molecules of pyruvate. The Krebs cycle occurs in the mitochondrial matrix. The Krebs cycle occurs in the mitochondrial matrix. –It degrades pyruvate to carbon dioxide. Several steps in glycolysis and the Krebs cycle transfer electrons from substrates to NAD +, forming NADH. Several steps in glycolysis and the Krebs cycle transfer electrons from substrates to NAD +, forming NADH. NADH passes these electrons to the electron transport chain. NADH passes these electrons to the electron transport chain. 9/1/20173

4. Phosphorylation I- Substrate-level phosphorylation: I- Substrate-level phosphorylation: Some ATP is generated in glycolysis and in Krebs cycle by Substrate-level phosphorylation. phosphate group is transferred from an organic molecule (the substrate) to ADP, forming 10% ATP (4 ATP). Some ATP is generated in glycolysis and in Krebs cycle by Substrate-level phosphorylation. phosphate group is transferred from an organic molecule (the substrate) to ADP, forming 10% ATP (4 ATP). II- Oxidative phosphorylation : II- Oxidative phosphorylation : As electrons passed along the chain, their energy stored in the mitochondrion in a form that can be used to synthesize the rest 90% of the ATP ATP (34 ATP). As electrons passed along the chain, their energy stored in the mitochondrion in a form that can be used to synthesize the rest 90% of the ATP ATP (34 ATP). via Oxidative phosphorylation. via Oxidative phosphorylation. Ultimately 38 ATP are produced per mole of glucose that is degraded to carbon CO 2 and H 2 O by respiration. Ultimately 38 ATP are produced per mole of glucose that is degraded to carbon CO 2 and H 2 O by respiration. 9/1/20174

5. General aspect A. More than 60% of our foods are carbohydrates. Starch, glycogen, sucrose, lactose and cellulose are the chief carbohydrates in our food. Before intestinal absorption, they are hydrolysed to hexose sugars (glucose, galactose and fructose). B. A family of a glycosidases that degrade carbohydrate into their monohexose components catalyzes hydrolysis of glycocidic bonds. These enzymes are usually specific to the type of bond to be broken. 9/1/20175

6. Digestion of carbohydrate by salivary α -amylase (ptylin) in the mouth: A. This enzyme is produced by salivary glands. Its optimum pH is 6.7. B. It is activated by chloride ions (cl-). C. It acts on cooked starch and glycogen breaking α 1-4 bonds, converting them into maltose [a disaccharide containing two glucose molecules attached by α 1-4 linkage]. This bond is not attacked by -amylase. Because both starch and glycogen also contain 1-6 bonds, the resulting digest contains isomaltose [a disaccharide in which two glucose molecules are attached by 1-6 linkage]. E. Because food remains for a short time in the mouth, digestion of starch and glycogen may be incomplete and gives a partial digestion products called: starch dextrins (amylodextrin, erythrodextrin and achrodextrin). F. Therefore, digestion of starch and glycogen in the mouth gives maltose, isomaltose and starch dextrins. 9/1/20176

7. III. ln the stomach: carbohydrate digestion stops temporarily due to the high acidity which inactivates the salivary - amylase. IV. Digestion of carbohydrate by the pancreatic - amylase small intestine in the small intestine. A. α -amylase enzyme is produced by pancreas and acts in small intestine. Its optimum pH is 7.1. B. It is also activated by chloride ions. C. It acts on cooked and uncooked starch, hydrolysing them into maltose and isomaltose. D. The final digestive processes occur at the small intestine and include the action of several disaccharidases. These enzymes are secreted through and remain associated with the brush border of the intestinal mucosal cells. 9/1/20177

8. B. The disaccharidases include: 1. Lactase ( β -galactosidase) which hydrolyses lactose into two molecules of glucose and galactose: Lactase Lactose Glucose + Galactose 2. Maltase ( α -glucosidase), which hydrolyses maltose into two molecules of glucose: Maltase Maltose Glucose + Glucose 3. Sucrose ( α -fructofuranosidase), which hydrolyses sucrose into two molecules of glucose and fructose: Sucrose Sucrose Glucose + Fructose 4. α - dextrinase (oligo-1,6 glucosidase) which hydrolyze (1,6) linkage of isomaltose. Dextrinase Isomaltose Glucose + Glucose 9/1/20178

9. VI. Digestion of cellulose: A. Cellulose contains β (1-4) bonds between glucose molecules. B. In humans, there is no β (1-4) glucosidase that can digest such bonds. So cellulose passes as such in stool. C. Cellulose helps water retention during the passage of food along the intestine  producing larger and softer feces  preventing constipation. 9/1/20179

10. Absorptions: introduction A.The end products of carbohydrate digestion are monosaccharides: glucose, galactose and fructose. They are absorbed from the jejunum to portal veins to the liver, where fructose and galactose are transformed into glucose. B.Two mechanisms are responsible for absorption of monosaccharides: active transport (against concentration gradient i.e. from low to high concentration) and passive transport (by facilitated diffusion). C. For active transport to take place, the structure of sugar should have: 1. Hexose ring. 2. OH group at position 2 at the right side. Both of which are present in glucose and galactose. Fructose, which does not contain -OH group to the right at position 2 is absorbed more slowly than glucose and galactose by passive diffusion (slow process). 3. A methyl or a substituted methyl group should be present at carbon 5. 9/1/201710

11. II. Mechanisms of absorption: A. Active transport: 1. Mechanism of active transport: a) In the cell membrane of the intestinal cells, there is a mobile carrier protein called sodium dependant glucose transporter (SGL T-1) It transports glucose to inside the cell using energy. The energy is derived from sodium-potassium pump. The transporter has 2 separate sites, one for sodium and the other for glucose. It transports them from the intestinal lumen across cell membrane to the cytoplasm. Then both glucose and sodium are released into the cytoplasm allowing the carrier to return for more transport of glucose and sodium. 9/1/201711

12. b) The sodium is transported from high to low concentration (with concentration gradient) and at the same time causes the carrier to transport glucose against its concentration gradient. The Na+ is expelled outside the cell by sodium pump. Which needs ATP as a source of energy. The reaction is catalyzed by an enzyme called "Adenosine triphosphatase (ATPase)". Active transport is much more faster than passive transport. c) Insulin increases the number of glucose transporters in tissues containing insulin receptors e.g. muscles and adipose tissue. 9/1/201712

13. 2. Inhibitors of active transport: a) Ouabin (cardiac glycoside): Inhibits adenosine triphosphatase (ATPase) necessary for hydrolysis of ATP that produces energy of sodium pump. b) Phlorhizin; Inhibits the binding of sodium in the carrier protein. B. Passive transport (facilitated diffusion): Sugars pass with concentration gradient i.e. from high to low concentration. It needs no energy. It occurs by means of a sodium independent facilitative transporter (GLUT -5). Fructose and pentoses are absorbed by this mechanism. Glucose and galactose can also use the same transporter if the concentration gradient is favorable. C. There is also sodium – independent transporter (GLUT-2), that is facilitates transport of sugars out of the cell i.e. to circulation. 9/1/201713

14. III. Defects of carbohydrate digestion and absorption: A. Lactase deficiency = lactose intolerance: 1. Definition: a) This is a deficiency of lactase enzyme which digest lactose into glucose and galactose b) It may be: (i) Congenital: which occurs very soon after birth (rare). (ii) Acquired: which occurs later on in life (common). 2. Effect: The presence of lactose in intestine causes: a) Increased osmotic pressure: So water will be drawn from the tissue (causing dehydration) into the large intestine (causing diarrhea). b) Increased fermentation of lactose by bacteria: Intestinal bacteria ferment lactose with subsequent production of CO2 gas. This causes distention and abdominal cramps. c) Treatment: Treatment of this disorder is simply by removing lactose (milk) from diet. 9/1/201714

15. B. Sucrose deficiency: A rare condition, showing the signs and symptoms of lactase deficiency. It occurs early in childhood. C. Monosaccharide malabsorption: This is a congenital condition in which glucose and galactose are absorbed only slowly due to defect in the carrier mechanism. Because fructose is not absorbed by the carrier system, its absorption is normal. IV. Fate of absorbed sugars: Monosaccharides (glucose, galactose and fructose) resulting from carbohydrate digestion are absorbed and undergo the following: A. Uptake by tissues (liver): After absorption the liver takes up sugars, where galactose and fructose are converted into glucose. B. Glucose utilization by tissues: Glucose may undergo one of the following fate: 9/1/201715

16. 1. Oxidation: through a) Major pathways (glycolysis and Krebs' cycle) for production of energy. b) Hexose monophosphate pathway: for production of ribose, deoxyribose and NADPH + H+ c) Uronic acid pathway, for production of glucuronic acid, which is used in detoxication and enters in the formation of mucopolysaccharide. 2. Storage: in the form of: a) Glycogen: glycogenesis. b) Fat: lipogenesis. 3. Conversion: to substances of biological importance: a) Ribose, deoxyribose  RNA and DNA. b) Lactose  milk. c) Glucosamine, galactosamine  mucopolysaccharides. d) Glucouronic acid  mucopolysaccharides. e) Fructose  in semen. 9/1/201716

17. Glucose Oxidation I. Glycolysis (Embden Meyerhof Pathway): A. Definition: 1. Glycolysis means oxidation of glucose to give pyruvate (in the presence of oxygen) or lactate (in the absence of oxygen). B. Site: cytoplasm of all tissue cells, but it is of physiological importance in: 1. Tissues with no mitochondria: mature RBCs, cornea and lens. 2. Tissues with few mitochondria: Testis, leucocytes, medulla of the kidney, retina, skin and gastrointestinal tract. 3. Tissues undergo frequent oxygen lack: skeletal muscles especially during exercise. 9/1/201717

18. C. Steps: Stages of glycolysis 1. Stage one (the energy requiring stage): a) One molecule of glucose is converted into two molecules of glycerosldhyde-3- phosphate. b) These steps requires 2 molecules of ATP (energy loss) 2. Stage two (the energy producing stage(: a) The 2 molecules of glyceroaldehyde-3-phosphate are converted into pyruvate (aerobic glycolysis) or lactate (anaerobic glycolysis(. b) These steps produce ATP molecules (energy production). D. Energy (ATP) production of glycolysis: ATP production = ATP produced - ATP utilized 9/1/201718

19. Glycolysis takes place in the cytosol of cells. Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially there is energy input corresponding to cleavage of two ~P bonds of ATP. 9/1/201719

20. 1. Hexokinase catalyzes: Glucose + ATP  glucose-6-P + ADP The reaction involves nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP. ATP binds to the enzyme as a complex with Mg ++. 9/1/201720

21. First Phase of Glycolysis The first reaction - phosphorylation of glucose Hexokinase or glucokinase This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous Be SURE you can interconvert K eq and standard state free energy change 9/1/201721

22. Hexokinase 1st step in glycolysis;  G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell K m for glucose is 0.1 mM; cell has 4 mm glucose So hexokinase is normally active! Glucokinase (K m glucose = 10 mM) only turns on when cell is rich in glucose Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - but is not the most important site of regulation of glycolysis - Why? 9/1/201722

23. Energy production of glycolysis: Net energyATP utilizedATP produced 2 ATP From glucose to glucose -6-p. From fructose -6-p to fructose 1,6 p. 4 ATP (Substrate level phosphorylation) 2ATP from 1,3 DPG. 2ATP from phosphoenol pyruvate In absence of oxygen (anaerobic glycolysis) 6 ATP Or 8 ATP 2ATP -From glucose to glucose -6-p. From fructose -6-p to fructose 1,6 p. 4 ATP (substrate level phosphorylation) 2ATP from 1,3 BPG. 2ATP from phosphoenol pyruvate. In presence of oxygen (aerobic glycolysis) + 4ATP or 6ATP (from oxidation of 2 NADH + H in mitochondria). 9/1/201723

24. E. oxidation of extramitochondrial NADH+H+: 1. cytoplasmic NADH+H+ cannot penetrate mitochondrial membrane, however, it can be used to produce energy (4 or 6 ATP) by respiratory chain phosphorylation in the mitochondria. 2. This can be done by using special carriers for hydrogen of NADH+H+ These carriers are either dihydroxyacetone phosphate (Glycerophosphate shuttle) or oxaloacetate (aspartate malate shuttle). a) Glycerophosphate shuttle: 1) It is important in certain muscle and nerve cells. 2) The final energy produced is 4 ATP. 3) Mechanism: - The coenzyme of cytoplasmic glycerol-3- phosphate dehydrogenase is NAD+. - The coenzyme of mitochodrial glycerol-3-phosphate dehydogenase is FAD. - Oxidation of FADH, in respiratory chain gives 2 ATP. As glycolysis gives 2 cytoplasmic NADH + H+  2 mitochondrial FADH, 2 x 2 ATP  = 4 ATP. b) Malate – aspartate shuttle: 1) It is important in other tissues patriculary liver and heart. 2) The final energy produced is 6 ATP. 9/1/201724

25. Differences between aerobic and anaerobic glycolysis: AnaerobicAerobic LactatePyruvate1. End product 2 ATP6 or 8 ATP2.energy Through Lactate formation Through respiration chain in mitochondria 3. Regeneration of NAD + Not available as lactate is cytoplasmic substrate Available and 2 Pyruvate can oxidize to give 30 ATP 4. Availability to TCA in mitochondria 9/1/201725

26. 2. Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose)  fructose-6-P (ketose) The mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure. A similar reaction catalyzed by Triosephosphate Isomerase will be presented in detail. 9/1/201726

27. 3. Phosphofructokinase catalyzes: fructose-6-P + ATP  fructose-1,6-bisP + ADP This highly spontaneous reaction has a mechanism similar to that of Hexokinase. The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated, as will be discussed later. 9/1/201727

28. Phosphoglucoisomerase Glucose-6-P to Fructose-6-P Why does this reaction occur?? ◦ next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH ◦ isomerization activates C-3 for cleavage in aldolase reaction Ene-diol intermediate in this reaction Be able to write a mechanism! 9/1/201728

29. 4. Aldolase catalyzes: fructose-1,6-bisphosphate  dihydroxyacetone-P + glyceraldehyde-3-P The reaction is an aldol cleavage, the reverse of an aldol condensation. Note that C atoms are renumbered in products of Aldolase. 9/1/201729

30. A lysine residue at the active site functions in catalysis. The keto group of fructose-1,6-bisphosphate reacts with the  -amino group of the active site lysine, to form a protonated Schiff base intermediate. Cleavage of the bond between C3 & C4 follows. 9/1/201730

31. 5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P  glyceraldehyde-3-P Glycolysis continues from glyceraldehyde-3-P. TIM's K eq favors dihydroxyacetone-P. Removal of glyceraldehyde-3-P by a subsequent spontaneous reaction allows throughput. 9/1/201731

32. The ketose/aldose conversion involves acid/base catalysis, and is thought to proceed via an enediol intermediate, as with Phosphoglucose Isomerase. Active site Glu and His residues are thought to extract and donate protons during catalysis. 9/1/201732

33. 9/1/201733

34. Glycolysis continued. Recall that there are 2 GAP per glucose. 9/1/201734

35. Balance sheet for ~P bonds of ATP:  How many ATP ~P bonds expended? ________  How many ~P bonds of ATP produced? (Remember there are two 3C fragments from glucose.) ________  Net production of ~P bonds of ATP per glucose: ________ 9/1/201735

36. They must reoxidize NADH produced in Glycolysis through some other reaction, because NAD + is needed for the Glyceraldehyde-3-phosphate Dehydrogenase reaction. Usually NADH is reoxidized as pyruvate is converted to a more reduced compound. The complete pathway, including Glycolysis and the reoxidation of NADH, is called fermentation. Fermentation: Anaerobic organisms lack a respiratory chain. 9/1/201736

37. E.g., Lactate Dehydrogenase catalyzes reduction of the keto in pyruvate to a hydroxyl, yielding lactate, as NADH is oxidized to NAD +. Lactate, in addition to being an end-product of fermentation, serves as a mobile form of nutrient energy, & possibly as a signal molecule in mammalian organisms. Cell membranes contain carrier proteins that facilitate transport of lactate. 9/1/201737

38. Skeletal muscles ferment glucose to lactate during exercise, when the exertion is brief and intense. Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted via Lactate Dehydrogenase back to pyruvate, which may be oxidized in Krebs Cycle or (in liver) converted to back to glucose via gluconeogenesis 9/1/201738

39. Lactate serves as a fuel source for cardiac muscle as well as brain neurons. Astrocytes, which surround and protect neurons in the brain, ferment glucose to lactate and release it. Lactate taken up by adjacent neurons is converted to pyruvate that is oxidized via Krebs Cycle. Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product. NADH is converted to NAD + in the reaction catalyzed by Alcohol Dehydrogenase. Glycolysis, omitting H + : glucose + 2 NAD + + 2 ADP + 2 P i  2 pyruvate + 2 NADH + 2 ATP Fermentation, from glucose to lactate: glucose + 2 ADP + 2 P i  2 lactate + 2 ATP Anaerobic catabolism of glucose yields only 2 “high energy” bonds of ATP. 9/1/201739

40. Substrate level phosphorylation: This means phosphorylation of ADP to ATP at the reaction itself.in glycolysis there are 2 examples: - 1.3 Bisphosphoglycerate + ADP 3 Phosphoglycerate + ATP - Phospho-enol pyruvate + ADP Enolpyruvate + ATP I. Special features of glycolysis in RBCs: 1. Mature RBCs contain no mitochondria, thus: a) They depend only upon glycolysis for energy production (=2 ATP). b) Lactate is always the end product. 2. Glucose uptake by RBCs is independent on insulin hormone. 3. Reduction of met-hemoglobin: Glycolysis produces NADH+H+, which used for reduction of met-hemoglobin in red cells. 9/1/201740

41. Biological importance (functions) of glycolysis: 1. Energy production: a) anaerobic glycolysis gives 2 ATP. b) aerobic glycolysis gives 8 ATP. 2. Oxygenation of tissues: Through formation of 2,3 bisphosphoglycerate, which decreases the affinity of Hemoglobin to O2. 3. Provides important intermediates: a) Dihydroxyacetone phosphate: can give glycerol-3phosphate, which is used for synthesis of triacylglycerols and phospholipids (lipogenesis). b) 3 Phosphoglycerate: which can be used for synthesis of amino acid serine. c) Pyruvate: which can be used in synthesis of amino acid alanine. 4. Aerobic glycolysis provides the mitochondria with pyruvate, which gives acetyl CoA Krebs' cycle. 9/1/201741

42. Reversibility of glycolysis (Gluconeoqenesis): 1. Reversible reaction means that the same enzyme can catalyzes the reaction in both directions. 2. all reactions of glycolysis -except 3- are reversible. 3. The 3 irreversible reactions (those catalyzed by kinase enzymes) can be reversed by using other enzymes. Glucose-6-p  Glucose F1, 6 Bisphosphate  Fructose-6-p Pyruvate  Phosphoenol pyruvate 4. During fasting, glycolysis is reversed for synthesis of glucose from non- carbohydrate sources e.g. lactate. This mechanism is called: gluconeogenesis. 9/1/201742

43. Comparison between glucokinase and hexokinase enzymes: HexokinaseGlucokinaase All tissue cellsLiver only1. Site High affinity (low km) i.e. it acts even in the presence of low blood glucose concentration. Low affinity (high km) i.e. it acts only in the presence of high blood glucose concentration. 2. Affinity to glucose Glucose, galactose and fructoseGlucose only3. Substrate No effectInduces synthesis of glucokinase.4. Effect of insulin Allosterically inhibits hexokinaseNo effect5.Effect of glucose-6-p It phosphorylates glucose inside the body cells. This makes glucose concentration more in blood than inside the cells. This leads to continuous supply of glucose for the tissues even in the presence of low blood glucose concentration. Acts in liver after meals. It removes glucose coming in portal circulation, converting it into glucose -6- phosphate. 6. Function 9/1/201743

44. Importance of lactate production in anerobic glycolysis: 1. In absence of oxygen, lactate is the end product of glycolysis: Glucose  Pyruvate  Lactate 2. In absence of oxygen, NADH + H + is not oxidized by the respiratory chain. 3. The conversion of pyruvate to lactate is the mechanism for regeneration of NAD +. 4. This helps continuity of glycolysis, as the generated NAD+ will be used once more for oxidation of another glucose molecule. 9/1/201744

45. As pyruvate enters the mitochondrion, a multienzyme complex modifies pyruvate to acetyl CoA which enters the Krebs cycle in the matrix. –A carboxyl group is removed as CO 2. –A pair of electrons is transferred from the remaining two-carbon fragment to NAD + to form NADH. –The oxidized fragment, acetate, combines with coenzyme A to form acetyl CoA. 9/1/201745

46. 9/1/201746