biochemistry

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

2. Glucose
 Roles of glucose
 Fuel (Glucose  CO2 + H2O ; ∆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 CO2
 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 CO2
 Electron-transfer reactions in mitochondria
 e- transfer to O2 to generate H2O
 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 CO2
 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+
 DG’1
o = -146 kJ/mol
 2ADP + 2Pi  2ATP + 2H2O
 DG’2
o = 2(30.5) = 61.0 kJ/mol
 Glc + 2NAD+ + 2ADP + 2Pi  2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O
 DG’s
o = DG’1
o + DG’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 DG‡ & increase reaction specificity
 Many glycolytic enzymes are specific for Mg2+ complexed with
phosphate groups

10. Glycolysis : Step 1
 1. Phosphorylation of Glc
 Hexokinase
 Substrates; D-glc & MgATP2-(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 DG’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, Zn2+ 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 DG’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+
 DG’o = -12.5 kJ/mol
 Coupling through 1,3-bisphophoglycerate
(common intermediate)
 Removal of 1,3-bisphosphoglycerate in
step 7  strong negative DG of step 6

19. Glycolysis : Step 8
 8. 3-phosphoglycerate to 2-
phosphoglycerate
 Phosphoglycerate mutase
 Mg2+
 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 + 2H2O
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 + Pi  Glc 1-P + (Glc)n-1
 Debranching enzyme
 Breakdown of (a16) branch
 Phosphoglucomutase
 Glc 1-P  Glc 6-P
 Bisphosphate intermediate

27. Digestion of Dietary Polysaccharides
and Disaccharides
 Digestion of starch and glycogen
 a-amylase in saliva
 Hydrolysis of starch to oligosaccharides
 Pancreatic a-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
 CO2 for brewing or baking
 Mg2+ & 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 B1 derivative
 Cleavage of bonds adjacent to a carbonyl group
 Decarboxylation of a-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 & CO2  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+ + 4H2O  Glc +
4ADP + 2GDP + 6Pi +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 DG 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 CO2 transfer  oxaloacetate
Pyruvate + HCO3
- + ATP  oxaloacetate + ADP + Pi

43. From Pyruvate to PEP
Oxaloacetate + GTP  PEP + CO2 + GDP
 PEP carboxykinase
 Cytosolic and mitochondria enzyme
 Overall reaction equation
 Pyruvate + ATP + GTP + HCO3
-
PEP + ADP + GDP + Pi + CO2, DG’o = 0.9 kJ/mol
But, DG = -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 : 105 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,
FADH2, 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 CO2 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