METABOLISM OF CARBOHYDRATES: GLYCOLYSIS

1. METABOLISM OF CARBOHYDRATES: GLYCOLYSIS For centuries, bakeries and breweries have exploited the conversion of glucose to ethanol and CO2 by glycolysis in yeast

2. DIGESTION OF CARBOHYDRATES Glycogen, starch and disaccharides(sucrose, lactose and maltose) are hydrolyzed to monosaccharide units in the gastrointestinal tract. The process of digestion starts in the mouth by the salivary enzyme –amilase. The time for digestion in mouth is limited. Salivary -amilaseis inhibited in stomach due to the action of hydrochloric acid. Another -amilaseis produced in pancreas and is available in the intestine.

3. -amilase -amilase hydrolyzes the -1-4-glycosidic bonds randomlyto produce smaller subunits like maltose, dextrines and unbranched oligosaccharides.

4. sucrase The intestinal juice contains enzymes hydrolyzing disaccharides into monosaccharides (they are produced in the intestinal wall) Sucrase hydrolyses sucrose into glucose and fructose Glucose Fructose Sucrose

5. lactase maltase Maltose Glucose Galactose Lactase hydrolyses lactose into glucose and galactose Lactose Glucose Glucose Maltase hydrolyses maltose into two glucose molecules

6. Protein Na+ Glucose ABSORPTION OF CARBOHYDRATES Only monosaccharides are absorbed The rate of absorption: galactose > glucose > fructose Glucose and galactose from the intestine into endothelial cells are absorbed by secondary active transport Protein

7. Carrier protein is specific for D-glucose or D-galactose. L-forms are not transported. There are competition between glucose and galactose for the same carrier molecule; thus glucose can inhibit absorption of galactose. Fructose is absorbed from intestine into intestinal cells by facilitated diffusion. Absorption of glucose from intestinal cells into bloodstream is by facilitated diffusion.

8. Transport of glucose from blood into cells of different organs is mainly by facilitated diffusion. The protein facilitating the glucose transport is called glucose transporter (GluT). GluT are of 5 types. GluT2 is located mainly in hepatocytes membranes (it transport glucose into cells when blood sugar is high); GluT1 is seen in erythrocytes and endothelial cells; GluT3 is located in neuronal cells (has higher affinity to glucose); GluT5 – in intestine and kidneys; GluT4 - in muscles and fat cells.

9. The fate of glucose molecule in the cell Glucose Pentose phosphate pathway supplies the NADPH for lipid synthesis and pentoses for nucleic acid synthesis Glycogenogenesis (synthesis of glycogen) is activated in well fed, resting state Glucose-6-phosphate Ribose, NADPH Glycogen Pyruvate Glycolysis is activated if energy is required

10. Glycolysis is the earliest discovered and most important process of carbohydrates metabolism. Glycolysis – metabolic pathway in which glucose is transformed to pyruvate with production of a small amount of energy in the form of ATP or NADH. Glycolysis is an anaerobic process (it does not require oxygen). Glycolysis pathway is used by anaerobic as well as aerobic organisms. In glycolysis one molecule of glucose is converted into two molecules of pyruvate. In eukaryotic cells,glycolysis takes place in the cytosol.

11. Acetyl CoA Pyruvate can be further metabolized to: (1)Lactate or ethanol (anaerobic conditions) (2) AcetylCoA (aerobic conditions) • Acetyl CoA is further oxidized to CO2 and H2O via the citric acid cycle • Much more ATP is generated from the citric acid cycle than from glycolysis

12. Catabolism of glucose in aerobic conditions via glycolysis and the citric acid cycle

13. The glycolytic pathway consist of ten enzyme-catalyzed reactions that begin with a glucose and split it into two molecules of pyruvate

14. Glycolysis (10 reactions) can be divided into three stages • In the 1st stage (hexosestage) 2 ATP are consumed per glucose • In the 3rd stage (triosestage)4 ATP are produced per glucose • Net: 2 ATP produced per glucose

15. Stage 1, which is the conversion of glucose into fructose 1,6-bisphosphate, consists of three steps: a phosphorylation, an isomerization, and a second phosphorylation reaction. The strategy of these initial steps in glycolysis is to trap the glucose in the cell and form a compound that can be readily cleaved into phospho-rylated three-carbon units.

16. Stage 2 is the cleavage of the fructose 1,6-bisphosphate into two three-carbon fragments dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Dihydroxyacetone phosphate and glyceraldehyde 3-phosphate are readily interconvertible.

17. In stage 3, ATP is harvested when the three-carbon fragments are oxidized to pyruvate.

18. Glycolysis Has 10 Enzyme-Catalyzed Steps 1. Hexokinase • Each chemical reaction prepares a substrate for the next step in the process • Transfers the g-phosphoryl of ATP to glucose C-6 oxygen to generate glucose 6-phosphate (G6P) • Four kinases in glycolysis: steps 1,3,7, and 10 • All four kinases require Mg2+ and have a similar mechanism

19. Properties of hexokinases • Broad substrate specificity - hexokinases can phosphorylate glucose, mannose and fructose • Isozymes- multipleforms of hexokinase occur in mammalian tissues and yeast • Hexokinases I, II, III are active at normal glucose concentrations • Hexokinase IV (Glucokinase) is active at higher glucose levels, allows the liver to respond to large increases in blood glucose • Hexokinases I, II and III are allosterically inhibited by physiological concentrations of their immediate product, glucose-6-phosphate, but glucokinase is not.

20. 2. Glucose 6-Phosphate Isomerase • Converts glucose 6-phosphate (G6P) (an aldose) to fructose 6-phosphate (F6P) (a ketose) • Enzyme preferentially binds the a-anomer of G6P (converts to open chain form in the active site) • Enzyme is highly stereospecific for G6P and F6P • Isomerase reaction is near-equilibrium in cells

21. 3. Phosphofructokinase-1 (PFK-1) • Catalyzes transfer of a phosphoryl group from ATP to the C-1 hydroxyl group of F6P to form fructose 1,6-bisphosphate (F1,6BP) • PFK-1 is metabolically irreversible and a critical regulatory point for glycolysis in most cells • A second phosphofructokinase (PFK-2) synthesizes fructose 2,6-bisphosphate (F2,6BP)

22. 4. Aldolase • Aldolase cleaves the hexose F1,6BP into two triose phosphates: glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) • Reaction is near-equilibrium, not a control point

23. 5. Triose Phosphate Isomerase (TPI) • Conversion of DHAP into GAP • Reaction is very fast, only the D-isomer of GAP is formed • Reaction is reversible. At equilibrium, 96% of the triose phosphate is DHAP. However, the reaction proceeds readily from DHAP to GAP because the subsequent reactions of glycolysis remove this product.

24. Fate of carbon atoms from hexose stage to triose stage

25. 6. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) • Conversion of GAP to 1,3-bisphosphoglycerate (1,3BPG) • Molecule of NAD+ is reduced to NADH • Energy from oxidation of GAP is conserved in acid-anhydride linkage of 1,3BPG • Next step of glycolysis uses the high-energy phosphate of 1,3BPG to form ATP from ADP

26. 7. Phosphoglycerate Kinase (PGK) • Transfer of phosphoryl group from the energy-rich mixed anhydride 1,3BPG to ADP yields ATP and 3-phosphoglycerate (3PG) • Substrate-level phosphorylation - Steps 6 and 7 couple oxidation of an aldehyde to a carboxylic acid with the phosphorylation of ADP to ATP

27. 8. Phosphoglycerate Mutase • Catalyzes transfer of a phosphoryl group from one part of a substrate molecule to another • Reaction occurs without input of ATP energy

28. 9. Enolase: 2PG to PEP • 2-Phosphoglycerate (2PG) is dehydrated to phosphoenolpyruvate (PEP) • Elimination of water from C-2 and C-3 yields the enol-phosphate PEP • PEP has a veryhigh phosphoryl group transfer potential because it exists in its unstable enol form

29. 10. Pyruvate Kinase (PK) PEP + ADP  Pyruvate + ATP • Catalyzes a substrate-level phosphorylation • Metabolically irreversible reaction • Regulation both by allostericmodulators and by covalentmodification • Pyruvate kinase gene can be regulated by various hormones and nutrients

30. Net reaction of glycolysis During the convertion of glucose to pyruvate: • Two molecules of ATP are produced • Two molecules of NAD+ are reduced to NADH Glucose + 2 ADP + 2 NAD+ + 2 Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

31. Scientific investigations into fermentation of grape sugar were pioneering studies of glycolysis GLYCOLYSIS

32. The Fate of Pyruvate The sequence of reactions from glucose to pyruvate is similar in most organisms and most types of cells. The fate of pyruvate is variable. Three reactions of pyruvate are of prime importance: 1. Aerobic conditions: oxidation to acetyl CoA which enters the citric acid cycle for further oxidation 2. Anaerobic conditions(muscles, red blood cells): conversion tolactate 3. Anaerobic conditions (microorganisms, yeast):conversion to ethanol

33. Diverse Fates of Pyruvate

34. Metabolism of Pyruvate to Ethanol Ethanolis formed from pyruvate in yeast andseveral other microorganisms in anaerobic conditions. Two reactions required: The first step is the decarboxylation of pyruvate to acetaldehyde. Enzyme - pyruvate decarboxylase. Coenzyme - thiamine pyrophosphate (derivative of the vitamin thiamine B1) The second step is the reduction of acetaldehyde to ethanol. Enzyme - alcohol dehydrogenase(active site contains a zinc). Coenzyme – NADH.

35. The conversion of glucose into ethanol is an example of alcoholic fermentation. The net result of alcoholic fermentation is: Glucose+2Pi + 2ADP + 2H+ 2ethanol + 2CO2 + 2ATP + 2H2O The ethanol formed in alcoholic fermentation provides a key ingredient for brewing and winemaking. There is no net NADH formation in the conversion of glucose into ethanol. NADH generated by the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of acetaldehyde to ethanol.

36. Metabolism of Pyruvate to Lactate Lactateis formed from pyruvate in an animal organism and in a variety of microorganisms in anaerobic conditions. The conversion of glucose into lactate is called lactic acid fermentation. Enzyme - lactate dehydrogenase. Coenzyme – NADH.

37. Muscles of higher organisms and humans lack pyruvate decarboxylase and cannot produce ethanol from pyruvate • Muscle contain lactate dehydrogenase. During intense activity when the amount of oxygen is limiting the lactic acid can be accumulated in muscles (lactic acidosis). • Lactate formed in skeletal muscles during exercise is transported to the liver. • Liver lactate dehydrogenase can reconvert lactate to pyruvate. Overall reaction in the conversion of glucose into lactate: Glucose + 2 Pi + 2 ADP  2lactate + 2 ATP + 2 H2O • As in alcoholic fermentation, there is no net NADH formation. • NADH formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate.

38. Metabolism of Pyruvate to Acetyl CoA In aerobic conditions pyruvate is converted to acetyl coenzyme A (acetyl CoA). Acetyl CoA enters citric acid cycle where degrades to CO2 and H2O and the energy released during such oxidation is utilized in NADH and FADH2. Pyruvate is converted to acetyl CoA in the matrix of mitochondria. The overall reaction: Pyruvate + NAD+ + CoA  acetyl CoA + CO2 + NADH Reaction is catalyzed by the pyruvate dehydrogenase complex (three enzymes and five coenzymes). If pyruvate is converted to acetyl CoA, NADH formed in the oxidation of glyceraldehyde 3-phosphate ultimately transfers its electrons to O2 through the electron-transport chain in mitochondria.

39. Other Sugars Can Enter Glycolysis • Glucoseis the main metabolic fuel in most organisms • Other sugars convert to glycolytic intermediates • Fructose and sucrose (contains fructose) are major sweeteners in many foods and beverages • Galactose from milk lactose (a disaccharide) • Mannose from dietary polysaccharides, glycoproteins

40. The Entry of Fructose into Glycolysis Much of the ingested fructose is metabolized by the liver, using the fructose 1-phosphate pathway. The first step is the phosphorylationof fructose to fructose 1-phosphate by fructokinase. Fructose 1-phosphate is then split into glyceraldehyde and dihydroxyacetone phosphate, an intermediate in glycolysis, by a specific fructose 1 -phosphate aldolase. Glyceraldehyde is then phosphorylated to glyceraldehyde 3-phosphate, a glycolytic intermediate, by triose kinase.

41. Fructose Is Converted to Glyceraldehyde 3-Phosphate

42. Fructose can be phosphorylated to fructose 6-phosphate by hexokinase. • However, the affinity of hexokinase for glucose is 20 times as great as it is for fructose. • Little fructose 6-phosphate is formed in the liver because glucose is so much more abundant in this organ. • Glucose, as the preferred fuel, is also trapped in the muscle by the hexokinase reaction. • Because liver and muscle phosphorylate glucose rather than fructose, adipose tissue is exposed to more fructose than glucose. • Hence, the formation of fructose 6-phosphate in the adipose tissue is not competitively inhibited to a biologically significant extent, and most of the fructose in adipose tissue is metabolized through fructose 6-phosphate.

43. The Entry of Galactose into Glycolysis Galactoseis converted into glucose 6-phosphatein four steps. The first reaction is the phosphorylation of galactose to galactose 1-phosphate by galactokinase.

44. Galactose 1-phosphate react with uridine diphosphate glucose (UDP-glucose). UDP-galactose and glucose 1-phosphate are formed. Enzyme - galactose 1-phosphate uridyl transferase. The galactose moiety of UDP-galactose is then epimerized to glucose. The configuration of the hydroxyl group at carbon 4 is inverted by UDP-galactose 4-epimerase.

46. Glucose 1-phosphate, formed from galactose, is isomerized to glucose 6-phosphate by phosphoglucomutase.

47. The Entry of Mannose into Glycolysis Mannoseis converted toFructose 6-Phosphate in two steps. Hexokinase catalyzes the convertion of mannose into mannose 6-phosphate. Isomerase converts mannose 6-phosphate into fructose 6-phosphate (metabolite of glycolysis).

48. Intolerance to Milk Many people are unable to metabolize the milk sugar lactose and experience gastro-intestinal disturbances if they drink milk. Lactose intolerance, or hypolactasia, is caused by a deficiency of the enzyme lactase, which cleaves lactose into glucose and galactose. Microorganisms in the colon ferment undigested lactose to lactic acid generating methane (CH4) and hydrogen gas (H2). The gas produced creates the uncomfortable feeling of gut distention and the annoying problem of flatulence. The lactic acid is osmotically active and draws water into the intestine, as does any undigested lactose, resulting in diarrhea. The gas and diarrhea hinder the absorption of other nutrients (fats and proteins). Treatment:- to avoid the products containing lactose; - the enzyme lactase can be ingested.

49. Galactosemia The disruption of galactose metabolism is referred to as galactosemia. Classic galactosemia is an inherited deficiency in galactose 1-phosphate uridyl transferase activity. Symptoms: - vomiting, diarrhea after consuming milk, - enlargement of the liver, jaundice, sometimes cirrhosis, - cataracts, - lethargy and retarded mental development, - markedly elevated blood-galactose level - galactose is found in the urine. The absence of the transferase in red blood cells is a definitive diagnostic criterion. The most common treatment is to remove galactose (and lactose) from the diet.

50. Regulation of Glycolysis The rate glycolysis is regulated to meet two major cellular needs: (1) the production of ATP, and (2) the provision of building blocks for synthetic reactions. There are three control sites in glycolysis - the reactions catalyzed by • hexokinase, • phosphofructokinase 1, and • pyruvate kinase These reactions are irreversible. Their activities are regulated • by the reversible binding of allostericeffectors • by covalent modification • by the regulation of transcription (change of the enzymes amounts). The time required for allosteric control, regulation by phosphorylation, and transcriptional control is typically in milliseconds, seconds, and hours, respectively.