1. Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

2. Glucose Roles of glucose  Fuel (Glucose  CO 2 + H 2 O ; ∆G = ~ -2,840 kJ/mol)  Precursor for other molecules Utilization of glucose in animals and plant  Synthesis of structural polymers  Storage  Glycogen, starch, or sucrose  Oxidation via glycolysis  Pyruvate for ATP and metabolic intermediate generations  Oxidation via pentose phosphate pathway  Ribose 5-P for nucleic acid synthesis  NADPH for reductive biosynthesis Generation of glucose  Photosynthesis : from CO 2  Gluconeogenesis (reversing glycolysis) : from 3-C or 4-C precursors

3. 14.1 Glycolysis Glycolysis Glucose 2 x Pyruvate 2 ATP & 2 NADH Fermentation the anaerobic degradation of glucose ATP production

4. An Overview: Glycolysis Two phases of glycolysis (10 steps)  Preparatory phase : 5 steps  From Glc to 2 glyceraldehyde 3-P  Consumption of 2 ATP molecules  Payoff phase : 5 steps  Generation of pyruvate  Generation of 4 ATP from high-energy phosphate compounds  1,3-bisphosphoglycerate, phosphoenylpyruvate  Generation of 2 NADH

5. Preparatory Phase

6. Payoff Phase

7. Fates of Pyruvate Aerobic conditions  Oxidative decarboxylation of pyruvate  Generation of acetyl-CoA  Citric acid cycle  Complete oxidation of acetyl-CoA  CO 2  Electron-transfer reactions in mitochondria  e - transfer to O 2 to generate H 2 O  Generation of ATP Fermentation : anaerobic conditions (hypoxia)  Lactic acid fermentation  Reduction of pyruvate to lactate  NAD + regeneration for glycolysis  Vigorously contracting muscle  Ethanol (alcohol) fermentation  Conversion of pyruvate to EtOH and CO 2  Microorganisms (yeast)

8. Fate of Pyruvate Anabolic fates of pyruvate  Source of C skeleton (Ala or FA synthesis)

9. ATP & NADH formation coupled to glycolysis Overall equation for glycolysis  Glc + 2 NAD +  2 pyruvate + 2NADH + 2H +   G’ 1 o = -146 kJ/mol  2ADP + 2Pi  2ATP + 2H 2 O   G’ 2 o = 2(30.5) = 61.0 kJ/mol  Glc + 2NAD + + 2ADP + 2Pi  2 pyruvate + 2NADH + 2H + + 2ATP + 2H 2 O   G’ s o =  G’ 1 o +  G’ 2 o = -85 kJ/mol  60% efficiency in conversion of the released energy into ATP Importance of phosphorylated intermediates  No export of phosphorylated compounds  Conservation of metabolic energy in phosphate esters  Binding energy of phosphate group  Lower  G ‡ & increase reaction specificity  Many glycolytic enzymes are specific for Mg 2+ complexed with phosphate groups

10. Glycolysis : Step 1 1. Phosphorylation of Glc  Hexokinase  Substrates; D -glc & MgATP 2- (ease nucleophilc attack by –OH of glc)  Induced fit  Soluble & cytosolic protein

11. Glycolysis : Step 2 2. Glc 6-P  Fru 6-P (isomerization)  Phosphohexose isomerase (phosphoglucose isomerase)  Reversible reaction (small  G’ o )

12. Glycolysis : Step 3 3. Phosphorylation of Fru 6-P to Fru 1,6-bisP  Phosphofructokinase-1 (PFK-1)  Irreversible, committed step in glycolysis  Activation under low [ATP] or high [ADP and AMP]  Phosphoryl group donor  ATP  PPi : some bacteria and protist, all plants

13. Glycolysi : Step 4 4. Cleavage of Fru 1,6-bisP  Dihydroxyacetone P & glyceraldehyde 3-P  Aldolase (fructose 1,6-bisphosphate aldolase)  Class I : animals and plant  Class II : fungi and bacteria, Zn 2+ at the active site  Reversible in cells because of lower concentrations of reactant

14. Class I Aldolase Reaction

15. Glycolysis : Step 5 5. Interconversion of the triose phosphates  Dihydroxyacetone P  glyceraldehyde 3-P  Triose phosphate isomerase

16. Glycolysis : Step 6 6. Oxidation of glyceraldehyde 3-P to 1,3- bisphosphoglycerate  Glyceraldehyde 3-P dehydrogenase  NAD + is the acceptor for hydride ion released from the aldehyde group  Formation of acyl phosphate  Carboxylic acid anhydride with phosphoric acid  High  G’ o of hydrolysis

17. Glyceraldehyde 3-P dehydrogenase

18. Glycolysis : Step 7 7. Phosphoryl transfer from 1,3- bisphosphoglycerate to ADP  3-phosphoglycerase kinase  Substrate-level phosphorylation of ADP to generate ATP  c.f. Respiration-linked phosphorylation Coupling of step 6 (endergonic) and step 7 (exergonic)  Glyceraldehyde 3-P + ADP + Pi + NAD +  3-phosphoglycerate + ATP + NADH + H +   G’ o = -12.5 kJ/mol  Coupling through 1,3-bisphophoglycerate (common intermediate)  Removal of 1,3-bisphosphoglycerate in step 7  strong negative  G of step 6

19. Glycolysis : Step 8 8. 3-phosphoglycerate to 2- phosphoglycerate  Phosphoglycerate mutase  Mg 2+  Two step reaction with 2,3-BPG intermediate

20. Glycolysis : Step 9 Dehydration of 2-phosphoglycerate to phosphoenolpyruvate (PEP)  Enolase  Free energy for hydrolysis  2-phosphoglycerate : -17.6 kJ/mol  PEP : -61.9 kJ/mol

21. Glycolysis : Step 10 Transfer of phosphoryl group from PEP to ADP  Pyruvate kinase  Substrate-level phosphorylation  Tautomerization from enol to keto forms of pyruvate  Irreversible  Important site for regulation

22. Overall Balance in Glycolysis Glucose + 2ATP + 2NAD + + 4ADP + Pi 2Pyruvate + 2ADP + 2NADH + 2H + + 4ATP + 2H 2 O Multienzyme complex Substrate channeling Tight regulation Rate of glycolysis: anaerobic condition (2ATP) aerobic condition (30-32) ATP consumption NADH regeneration Allosteric regulation of enzymes; Hexokinase, PFK-1, pyruvate kinase Hormone regulations; glucagon, insulin, epinephrine Changes in gene expression for the enzymes

24. 14.2 Feeder Pathways for Glycolysis

25. Entry of Carbohydrates into Glycolysis

26. Degradation of Glycogen and Starch by Phosphorolysis Glycogen phosphorylase  (Glc) n + P i  Glc 1-P + (Glc) n-1 Debranching enzyme  Breakdown of (  1  6) branch Phosphoglucomutase  Glc 1-P  Glc 6-P  Bisphosphate intermediate

27. Digestion of Dietary Polysaccharides and Disaccharides Digestion of starch and glycogen   -amylase in saliva  Hydrolysis of starch to oligosaccharides  Pancreatic  -amylase   maltose and maltotriose, limit dextrin Hydrolysis of intestinal dextrins and disaccharides  Dextrinase  Maltase  Lactase  Sucrase  Trehalase Transport of monosaccharide into the epithelial cells c.f. lactase intolerance  Lacking lactase activity in the intestine  Converted to toxic product by bacteria  Increase in osmolarity  increase in water retention in the intestine

28. Entry of Other monosaccharides into Glycolytic Pathway Fructose  In muscle and kidney  Hexokinase  Fru + ATP  Fru 6-P + ADP  In liver  Fructokinase  Fru + ATP  Fru 1-P + ADP  Fructose 1-P aldolase Glyceraldehyde 3-P Triose phosphate isomerase Triose kinase

29. Galactose  Glactokinase; Gal  Glc 1-P  Galatosemia  Defects in the enzymatic pathway Mannose  Hexokinase  Man + ATP  Man 6-P + ADP  Phosphomannose isomerase  Man 6-P  Fru 6-P Entry of Other monosaccharides into Glycolytic Pathway

30. 14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation

31. Pyruvate fates Hypoxic conditions - Rigorously contracting muscle - Submerged plant tissues - Solid tumors - Lactic acid bacteria Failure to regenerate NAD + Fermentation is the way of NAD + regeneration

32. Lactic Acid Fermentation Lactate dehydrogenase  Regeneration of NAD +  Reduction of pyruvate to lactate Fermentation  No oxygen consumption  No net change in NAD + or NADH concentrations  Extraction of 2 ATP

33. Ethanol Fermentation Two step process Pyruvate decarboxylase  Irreversible decarboxylation of pyruvate  Brewer’s and baker’s yeast & organisms doing ethanol fermentation  CO 2 for brewing or baking  Mg 2+ & thiamine pyrophosphate (TPP) Alcohol dehydrogenase  Acetaldehyde + NADH + H +  EtOH + NAD +  Human alcohol dehydrogenase  Used for ethanol metabolism in liver

34. Thiamine Phyrophosphate (TPP) as Active Aldehyde Group Carrier TPP  Vitamin B 1 derivative  Cleavage of bonds adjacent to a carbonyl group  Decarboxylation of  -keto acid  Rearrangement of an activated acetaldehyde group

35. Role of Thiamine Pyrophosphate (TPP) in pyruvate decarboxylation TPP  Nucleophilic carbanion of C-2 in thiazolium ring  Thiazolium ring acts as “e - sink”

36. Fermentation in Industry Food  Yogurt  Fermentation of carbohydrate in milk by Lactobacillus bulgaricus  Lactate  low pH & precipitation of milk proteins  Swiss cheese  Fermentation of milk by Propionibacterium freudenreichii  Propionic acid & CO 2  milk protein precipitation & holes  Other fermented food  Kimchi, soy sauce  Low pH prevents growth of microorganisms Industrial fermentation  Fermentation of readily available carbohydrate (e.g. corn starch) to make more valuable products  Ethanol, isopropanol, butanol, butanediol  Formic, acetic, propionic, butyric, succinic acids

37. 14.4 Gluconeogenesis

38. Gluconeogenesis Pyruvate & related 3-/ 4-C compounds  glucose Net reaction  2 pyruvate + 4ATP + 2GTP + 2NADH + 2H + + 4H 2 O  Glc + 4ADP + 2GDP + 6P i +2NAD + In animals  Glc generation from lactate, pyruvate, glycerol, and amino acids  Mostly in liver  Cori cycle ; Lactate produced in muscle  converted to glc in liver  glycogen storage or back to muscle In plant seedlings  Stored fats & proteins  disaccharide sucrose In microorganisms  Glc generation from acetate, lactate, and propionate in the medium

39. Gluconeogenesis

40. Glycolysis vs. Gluconeogenesis 7 shared enzymatic reactions 3 bypass reactions; irreversible steps requiring unique enzymes  Large negative  G in glycolysis  Hexokinase vs. glc 6-phosphatase  Phosphofructokinase-1 vs. fructose 1,6-bisphosphatase  Pyruvate kinase vs. pyruvate carboxylase + PEP carboxykinase

41. From Pyruvate to PEP Pyruvate carboxylase  Mitochondrial enzyme with biotin coenzyme  Activation of pyruvate by CO 2 transfer  oxaloacetate Pyruvate + HCO 3 - + ATP  oxaloacetate + ADP + P i

43. From Pyruvate to PEP Oxaloacetate + GTP  PEP + CO 2 + GDP PEP carboxykinase  Cytosolic and mitochondria enzyme Overall reaction equation  Pyruvate + ATP + GTP + HCO 3 - PEP + ADP + GDP + P i + CO 2,  G’ o = 0.9 kJ/mol But, G = -25 kJ/mol

44. Alternative paths from pyruvate to PEP From pyruvate  Oxaloacetate + NADH + H +  malate + NAD + (mitochondria)  Malate + NAD +  oxaloacetate + NADH + H + (cytosol)  [NADH]/[NAD + ] in cytosol : 10 5 times lower than in mitochondria  Way to provide NADH for gluconeogenesis in cytosol From lactate  NADH generation by oxidation of lactate  No need to generate malate intermediate

45. 14.5 Pentose Phosphate Pathway of Glucose Oxidation

46. Pentose Phosphate Pathway Oxidative phase; NADPH & Ribose 5-P Nonoxidative phase  Recycling of Ribulose 5-P to Glc 6-P  Pentose ribose 5-phosphate  Synthesis of RNA/DNA, ATP, NADH, FADH 2, coenzyme A in rapidly dividing cells (bone marrow, skin etc)  NADPH  Reductive biosynthesis - Fatty acid (liver, adipose, lactating mammary gland) - Steroid hormones & cholesterol (liver, adrenal glands, gonads)  Defense from oxygen radical damages - High ratio of NADPH/NADP +  a reducing atmosphere  preventing oxidative damages of macromolecules

47. Oxidative Pentose Phosphate Pathway

48. Nonoxidative Pentose Phosphate Pathway 6 Pentose phosphates  5 Hexose phosphates Reductive pentose phosphate pathway  Reversal of nonoxidative Pentose Phosphate Pathway  Photosynthetic assimilation of CO 2 by plant

49. Nonoxidative Pentose Phosphate Pathway Transketolase  Transfer of a 2-C fragment from a ketose donor to an aldose acceptor  Thiamine pyrophosphate (TPP) cofactor Transaldolase  Transfer of a 3-C fragment  Lys : Schiff base with the carbonyl group of ketose Stabilization of carbanion intermdeidate

50. Nonoxidative Pentose Phosphate Pathway

51. Regulation of Pentose phosphate Pathway