Carbohydrate Metabolism

1. Carbohydrate Metabolism Prof. Omar Al-Attas Biochemistry Department College of Science, King Saud University

2. Function of Metabolism • Synthesis and degrade biomolecules. • Obtain energy from the environment. • Convert it into cell’s own characteristic molecule. • Polymerize monomeric precursors into macro molecules.

3. Strategy of metabolism The basic strategic Is to for ATP, reducing power and building block for biosynthesis • ATP is the universal currency of energy. • ATP is generated by the oxidation of fuel molecules. • NADPH electron donor • Construction of biomolecules from relative small building blocks. • Biosynthesis and degradative pathways are distinct.

4. Carbohydrates • Compounds made of carbons, hydrogen and oxygen. • General formula is CnH2nOn. • Principal source of energy in the organism. • The central molecule is Glucose

5. Metabolic is controlled in several ways: • Allosteric modifiers: Enzyme catalyzing non-equilibrium reaction are often allosteric proteins subject to the rapid action of “feed –back” or “feed-forward” control by allosteric modifiers. Also, the flow of molecules is determined by the amount and activities of certain enzymes rather than by the amount of substrate available. The first irreversible in a pathway (the committed step) is usually an important control element

6. Cont…. Generally, for an allostericenzyme, rate(v) vs [S] is Sigmoidalinstead of hyperbolic. v sigmoidal [S] • Simple enzyme = hyperbolic: at low [S], activity fairly high • Complex, allosteric = sigmoidal: activity low • at low [S], increases greatly in midrange • Efficiency is better, more responsive to [S]

7. Cont… 2. Covalent modification some regulatory enzymes are controlled by covalent modification i.e. phosphorylation and dephophorilation. This is rapid and often mediated through the formation of cAMP, which in turn causes the conversion of an inactive enzyme into an active enzyme. Example of phosphorylation: the enzyme phosphorylase a in glycogen utilization glycogen = polymer of glucose in muscle & liver. glycogen function: store & release glucose.

8. Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi Energy source Enzyme for removing glucose-1-phosphate is phosphorylase a, activated by phosphorylation. Another & another & another glu-1-Pi is removed Part of cAMP cascade (Pi = inorganic phosphate)

9. Cont… 3. Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors 4. Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartments. Glycolysis, Pentose P pathway, FA sythesis take place in the cytosol. FA oxidation, TCA cycle, oxydative decarboxylation in mitochrondria Gluconeogenesis, urea cycle synthesis in both compartment

10. Cont… 5. Metabolic specialization of Organs: Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

11. 6. Mass Action Ration Cont… • Is determined by the rate-controlling steps in a pathway by measuring how close the reactions are to equilibrium. • Mass action ration: Products/substrates (measured in the intact cells) • If the reaction is close to equilibrium , the mass action ration will be close to the equilibrium constant, If however, the reaction is rate controlling in the cell, it will not approach equilibrium and the mass action ration will be lower than equilibrium constant.

12. Control of Substrate By Insulin Insulin • Hormone produce by the beta cells in the pancreas. • Stored as proinsulin (inactive form) as small granules. • Its release is triggered by increased glucose levels in the blood. • Stimulates glucose uptake by tissue by binding to receptors in the cell membrane. Permits glucose to enter cell.

13. Glycolysis • First stage of carbohydrate catabolism. • Is an anaerobic process. • Simple sugar are broken into pyruvate. • All organism uses this process Overall it uses 1 molecule of glucose, 2 ADP, 2 ATP, 2 NAD+, and 2 PO4, and 10 different enzymes. • The net energy production is about 96000 calorie or 8 ATP. 6 carbon stage Requires energy

14. Reaction of Glycolysis 3 carbon stage Double this since 2 pyruvate are made

15. Control of Glucose levels

16. Glycogenolysis: Breakdown of glycogen Glucagon • This hormone is also produced in the pancreas in an inactive form. • Low glucose levels results in it conversion to an active form and its release. • Its entry into the liver cells result in the conversion of glycogen to glucose, with glucose being released to the blood.

17. Glycogen Synthesis • It is known as glycogenesis. • Synthesis from glucose occurs. • It is carried out by the enzyme glycogen synthesis. • Occurs when there is excess of glucose in the body thus helps in the control of glucose in the blood. • Is stored in the liver. • Occurs in all the tissue of the body but the major sites are liver and muscle.

18. Regulation of Glycolysis • Glycolysis is a sequence in the cytosol which converts

19. Cont… • The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADH • It is also called as the Embden-Meyerhof Pathway • Glycolysis is a universal pathway; present in all organisms: from yeast to mammals. • In eukaryotes, glycolysis takes place in the cytosol • Glycolysis is anaerobic; it does not require oxygen • In the presence of O2, pyruvate is further oxidized to CO2. In the absence of O2, pyruvate can be fermented to lactate or ethanol. • Net Reaction: Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 pyruvate + 2 ATP + 2 NADH + 2 H2O The 3 stages of Glycolysis

20. The 3 stages of Glycolysis • Stage 1 is the investment stage. 2 mols of ATP are consumed for each mol of glucose • Glucose is converted to fructose-1,6-bisphosphate. • Glucose is trapped inside the cell and at the same time converted to an unstable form that can be readily cleaved into 3-carbon units. • In stage 2 fructose-1,6-bisphosphate is cleaved into 2 3- carbon units of glycerladehyde-3-phosphate. • Stage 3 is the harvesting stage. 4 mols of ATP and 2 mols of NADH are gained from each initial mol of glucose. This ATP is a result of substrate-level phosphorylation • Glyceraldehyde-3-phosphate is oxidized to pyruvate

21. Step-wise reactions of glycolysis • Reaction 1: Phosphorylation of glucose to glucose-6 phosphate. • This reaction requires energy and so it is coupled to the hydrolysis of ATP to ADP and Pi. • Enzyme: hexokinase. It has a low Km for glucose; thus, once glucose enters the cell, it gets phosphorylated. • This step is irreversible. So the glucose gets trapped inside the cell. (Glucose transporters transport only free glucose, not phosphorylated glucose) • Reaction 2: Isomerization of glucose-6-phosphate to fructose 6- phosphate. The aldose sugar is converted into the keto isoform. • Enzyme: phosphoglucomutase. • This is a reversible reaction. The fructose-6-phosphate is quickly consumed and the forward reaction is favored.

22. Cont… Reaction 3: is another kinase reaction. Phosphorylation of the hydroxyl group on C1 forming fructose-1,6- bisphosphate. • Enzyme: phosphofructokinase. This allosteric enzyme regulates the pace of glycolysis. • Reaction is coupled to the hydrolysis of an ATP to ADP and Pi. • This is the second irreversible reaction of the glycolytic pathway. • Reaction 4: fructose-1,6-bisphosphate is split into 2 3-carbon molecules, one aldehyde and one ketone: dihyroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). • The enzyme is aldolase. • Reaction 5: DHAP and GAP are isomers of each other and can readily inter-convert by the action of the enzyme triose-phosphate isomerase. • GAP is a substrate for the next step in glycolysis so all of the DHAP is eventually depleted. So,

23. Cont… • Upto this step, 2 molecules of ATP were required for each molecule of glucose being oxidized • The remaining steps release enough energy to shift the balance sheet to the positive side. This part of the glycolytic pathway is called as the payoff or harvest stage. • Since there are 2 GAP molecules generated from each glucose, each of the remaining reactions occur twice for each glucose molecule being oxidized. • Reaction 6: GAP is dehydrogenated by the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In the process, NAD+ is reduced to NADH + H+ from NAD. Oxidation is coupled to the phosphorylation of the C1 carbon. The product is 1,3-bisphosphoglycerate.

24. Cont… Reaction 7: BPG has a mixed anhydride, a high energy bond, at C1. This high energy bond is hydrolyzed to a carboxylic acid and the energy released is used to generate ATP from ADP. Product: 3-phosphoglycerate. Enzyme: phosphoglycerate kinase. • Reaction 8: The phosphate shifts from C3 to C2 to form 2- phosphoglycerate. Enzyme:phosphoglycerate mutase. • Reaction 9: Dehydration catalyzed by enolase (a lyase). A water molecule is removed to form phosphoenolpyruvate which has a double bond between C2 and C3. • Reaction 10: Enolphosphate is a high energy bond. It is hydrolyzed to form the enolic form of pyruvate with the synthesis of ATP. The irreversible reaction is catalyzed by the enzyme pyruvate kinase. Enol pyruvate quickly changes to keto pyruvate which is far more stable.

25. Glycolysis: Energy balance sheet • Hexokinase: - 1 ATP • Phosphofructokinase: -1 ATP • GAPDH: +2 NADH • Phsophoglycerate kinase: +2 ATP • Pyruvate kinase: +2 ATP Total/ molecule of glucose: +2 ATP, +2 NADH

26. Fate of Pyruvate • NADH is formed from NAD+ during glycolysis. • The redox balance of the cell has to be maintained for further cycles of glycolysis to continue. • NAD+ can be regenerated by one of the following reactions /pathways: • Pyruvate is converted to lactate • Pyruvate is converted to ethanol • In the presence of O2, NAD+ is regenerated by ETC. Pyruvate is converted to acetyl CoA which enters TCA cycle and gets completely oxidized to CO2.

27. Lactate Fermentation Formation of lactate catalyzed by lactate dehydrogenase: CH3-CO-COOH + NADH + H+􀃅􀃆 CH3-CHOH-COOH + NAD+ • In highly active muscle, there is anaerobic glycolysis because the supply of O2 cannot keep up with the demand for ATP. • Lactate builds up causing a drop in pH which inactivates glycolytic enzymes. End result is energy deprivation and cell death; the symptoms being pain and fatigue of the muscle. • Lactate is transported to the liver where it can be reconverted to pyruvate by the LDH reverse reaction

28. Ethanol fermentation • Formation of ethanol catalyzed by 2 enzymes • Pyruvate decarboxylase catalyzes the first irreversible reaction to form acetaldehyde: CH3-CO-COOH 􀃆 CH3-CHO + CO2 • Acetaldehyde is reduced by alcohol dehydogenase is a reversible reaction: CH3-CHO + NADH + H+ 􀃅􀃆 CH3CH2OH + NAD+ • Ethanol fermentation is used during wine-making

29. Cont… Fructose is phosphorylated by fructokinase (liver) or hexokinase (adipose) on the 1 or 6 positions resp. • Fructose-6-phosphate is an intermediate of glycolysis. • Fructose-1-phosphate is acted upon by an aldolase-like enz that gives DHAP and glyceraldehyde. • DHAP is a glycolysis intermediate and glyceraldehyde can be phosphorylated to glyceraldehyde-3-P. • Glycerol is phosphorylated to G-3-P which is then converted to glyceraldehyde 3 phosphate. • Galactose has a slightly complicated multi-step pathway for conversion to glucose-1-phosphate. • gal 􀃆 gal-1-P 􀃆 UDP-gal 􀃆 UDP-glc 􀃆 glc-1-P. • If this pathway is disrupted because of defect in one or more enz involved in the conversion of gal to glc-1-P, then galactose accumulates in the blood and the subject suffers from galactosemia which is a genetic disorder, an inborn error of metabolism.

30. The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADH • It is also called as the Embden-Meyerhof Pathway • Glycolysis is a universal pathway; present in all organisms: from yeast to mammals. • In eukaryotes, glycolysis takes place in the cytosol • Glycolysis is anaerobic; it does not require oxygen • In the presence of O2, pyruvate is further oxidized to CO2. In the absence of O2, pyruvate can be fermented to lactate or ethanol. • Net Reaction: Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 pyruvate + 2 ATP + 2 NADH + 2 H2O

31. The citric acid cycle 1. Final stage for the metabolism of carbohydrates, fats and amino acids 2. Oxidative cycle 3. Requires oxygen 4. Aerobic 5. Also called the Krebs cycle for Hans Krebs who first described it. • Citric acid cycle: • Also known as tricarboxylic acid cycle (TCA cycle) or the Krebs's cycle. • Pyruvate formed after glycolysis undergoes oxydativedecarboxylation to form acytyl coenzyme A. • The acetyl co A gets completely oxidzed into CO2 by TCA cycle.

32. Control of Citric acid cycle • Several route for controlling the cycle. • Insufficient Oxygen. • Reduced energy demand causes a build up of ATP, NADH inhibits –pyruvate conversion to acetyl CoA conversion. Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle • Excess ADP will stimulate many of these steps to things go faster

33. The Citric Acid Cycle • In aerobic organisms, pyruvate (formed through glycolysis) oxidized to CO2 and acetyl-CoA using coenzyme A • Subsequent oxidation of acetyl group carried out using the citric acid cycle • Citric acid cycle is amphibolic • Catabolic and anabolic • Aerobic catabolism of carbohydrates, lipids, and amino acids merge at citric acid cycle • Oxidized acetyl-CoA formed from metabolism of all three • Intermediates of citric acid cycle are starting points for many biosynthetic pathways

34. In Cytosol In Mitochondria

35. The amphibolic Citric Acid Cycle

36. cont… • Controlled metabolic oxidation • Compounds oxidized in discrete steps • Enzymatic transfer of electrons to O2 • Analogous to combustion of organic compounds to produce CO2 and H2O and heat • Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH and ubiquinol (QH2) • Re-oxidation of reduced coenzymes produce more energy (ATP) through electron transport and oxidative phosphorylation • aka: • tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates) • Krebs cycle (for biochemist Hans Krebs)

37. Net reaction • Circular pathway • Regenerate oxaloacetate starting material in last step of cycle • Cycle is a multi-step enzyme, can catalyze unlimited acetyl groups • Carbon flow: • 2 carbons that enter the cycle are not the same carbons lost as CO2 • Series of 8 enzyme-catalyzed reactions • Transfer of 4 pairs of electrons • Production of energy-rich molecules: • Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2) • Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation • 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2 • Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell • Net reaction: Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → 2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

38. The pathway

39. Synthesis of acetyl-CoA • Not part of cycle, but must occur first • First step: entry of pyruvate into the mitochondrian • In aerobic cells, all enzymes of the citric acid cycle are located within the mitochondrion • Mitochondrion enclosed by a double membrane • Pyruvate passes through outer membrane via aqueous channels formed by transmembrane proteins (porins) • Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

40. Conversion of pyruvate to acetyl-CoA • Oxidative decarboxylation • Series of 5 reactions • Irreversible • Mechanism is highly complicated • Catalyzed by complex of enzymes and cofactors • Pyruvate dehydrogenase complex • Multi-enzyme structure located in mitochondrial matrix • Contains multiple copies of three non-covalently associated enzymes and five coenzymes • E1 and E3 surround core of 24-60 E2 chains (# chains depends on type of cell) • Overall reaction: CH3C(O)CO2- + NAD+ + CoASH → CH3C(O)-SCoA + NADH + CO2

41. 1. Formation of citrate • Oxaloacetate reacts with acetyl-CoA to form citrate and coenzyme A • Aldol condensation • Only C-C bond-forming reaction in cycle • Irreversible • Enzyme = citrate synthase

42. Citrate synthase • Dimer of two identical subunits • Changes in conformation • Binding of oxaloacetate • Domains move closer to form binding site for acetyl-CoA • Formation of intermediate • Enzyme closes around intermediate • Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent • Intermediate hydrolyzed by bound water molecule • Enzyme opens and products leave active site

43. 2. Isomerization of citrate to isocitrate • Citrate is a 3° alcohol • Cannot be oxidized to keto acid • Isocitrate is a 2° alcohol • Easily oxidized • Mechanism: • First step: elimination of H2O to from alkene intermediate (cis-aconitate) • Second step: stereospecific addition of water to form (2R, 3S)-isocitrate • Reaction near equilibrium • Enzyme = aconitase • aka aconitate hydratase • Named for intermediate • Binds C3 carboxylate and hydroxyl groups • Substrate positioning essential for stereospecificity

44. 3. Oxidative decarboxylation of isocitrate to form a-ketoglutarate • First of four oxidation-reduction reactions • NAD+ is oxidizing agent • Mechanism: • First step: alcohol oxidized by transfer of H:- from C2 to NAD+ • Intermediate = oxalosuccinate, an unstable b-keto acid • First molecule of NADH formed • Second step: intermediate undergoes b-decarboxylation to form an a-keto acid, which is released from enzyme • First molecule of CO2 produced • Irreversible • One of rate-limiting steps in cycle • Enzyme = isocitrate dehydrogenase

45. 4. Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA • Catalyzed by multi-enzyme a-ketoglutarate dehydrogenase complex • a-ketoglutarate dehydrogenase (E1) • Dihydrolipoamide succinyltransferase (E2) • Dihyrdolipoamide dehydrogenase (E3) • Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex • Same coenzymes • Similar complicated mechanism • Product is high-energy thioester • Key regulatory step of citric acid cycle • Second molecule of NADH produced • Second molecule of CO2 produced

46. Halfway through the cycle… • So far… • Net oxidation of two carbon atoms to produce two molecules CO2 • In the next four reactions… • Four-carbon succinyl group of succinyl CoA converted back to oxaloacetate • As oxaloacetate is regenerated, additional acetyl- CoA enters the citric acid cycle to be oxidized

47. 5. Conversion of succinyl-CoA to succinate • Substrate-level phosphorylation • Cleavage of high-energy thioester bond • Free energy conserved through the synthesis of nucleoside triphosphate • GTP in mammals • ATP in plants and bacteria • GDP regenerated and ATP produced from the reaction of GTP with ADP • GTP + ADP GDP + ATP • Nucleoside diphosphate kinase • Enzyme = succinyl-CoA synthetase • aka succinate thiokinase • Mechanism: • Phosphate displace CoA from bound succinyl-CoA molecule • Phosphoryl group transfers to His residue of enzyme • Succinate released • Phosphoryl group transferred to GDP (or ADP)

48. Succinyl-CoA synthetase mechanism

49. 6. Oxidation of succinate to fumarate • Dehydrogenation (loss of H2; oxidation) • Stereospecific to form trans double bond only • Catalyzed by succinate dehydrogenase complex • aka succinate dehydrogenase • aka Complex II • Embedded in inner mitochondrial membrane, rather than in mitochondrial matrix • Oxidation of alkane requires stronger oxidizing agent than NAD+ (hence FAD) • FADH2 produced is re-oxidized by coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2) • Competitive inhibitor = malonate • -O2C-CH2-CO2- • Binds to active site through carboxylate groups • Cannot undergo dehydrogenation • Inhibition reactions used by Krebs to determine citric acid cycle reaction sequence • Symmetrical molecule evenly distributes carbons in remainder of products throughout the cycle

50. 7. Hydration of fumarate to form L-malate • Reversible reaction, near equilibrium • Stereospecificity • trans addition of water to double bond of fumarate • Only trans double bond will react • Enzyme = fumarase • aka fumarate hydratase