1. GLYCOLYSIS
Definition: from Greek “glykys” (sweet) & “lysis” (splitting)

2. “Living organisms, like machines, conform to the law of the conservation of energy, and must pay for all their activities in the currency of catabolism”
Ernest Baldwin, Dynamic Aspects of Biochemistry (1952)

3. I. BACKGROUND Glycolysis Carried out by nearly every living cell
In cytosol of eukaryotes
Catabolic process
Releases energy stored in covalent bonds
Stepwise degradation
Glucose
Other simple sugars

4. I. Background, cont… Anaerobic process
Evolved in an environment lacking O2
Primitive earth … millions of years ago
Early, important pathway
Provided means to extract energy from nutrient molecules
Central role in anaerobic metabolism
For the first 2 billion years of biological evolution on earth
Modern organisms
Provides precursors for aerobic catabolic pathways
Short term anaerobic energy source

5. Supplies metabolic intermediates Three fates Storage
Background, cont…
Glucose is a precursor
Supplies metabolic intermediates
Three fates
Storage
Oxidation to pyruvate
Oxidation to pentoses

7. beta D-Glucose is the major fuel Rich in potential energy
Background, cont…
beta D-Glucose is the major fuel
Rich in potential energy
Stored in bonds
Is literally solar energy
ΔG01= kJ/mole
Advantages to glucose
Catabolism  ATP
Can be stored
Eg: Polysaccarides, sucrose
Can be transported
Blood glucose
Organism to organism

8. History Background, cont… Began with Pasteur: Mid- nineteenth century
Eduard Buchner: 1897
Fermentation in broken extracts of yeast cells
Arthur Harden and William Young: 1905
Discover phosphate is required for glucose fermentation
Gustov Embden, Otto Meyerhof and Jocob Parnas
Seminal work
Often called the Embden-Meyerhof-Parnas pathway
Elucitated in 1940
Fritz Lipmann and Herman Kalckar: 1941
Metabolic role of high energy compounds like ATP

9. II. GLYCOLYSIS “Most completely understood biochemical pathway”
Sequence of 10 enzymatic pathways
1 molecule of glucose is converted to 2 3-carbon pyruvate molecules
Concomitant generation of 2 ATP
Key role in energy metabolism
Provides free energy for organisms
Prepares glucose (and other molecules) for further oxidative degradation

10. Function, Glycolysis, cont…
Most carbon in cells follows this pathway
Only source of energy for many tissues
Rates and Regulation vary among species
Most significant difference is the way that pyruvate is utilized

11. The fates of pyruvate Aerobic Anaerobic Glycolosis, cont…
Oxidative decarboxylation to acetyl
2-cabon molecule
Forms acetyl-coenzyme A
To Krebs cycle
Electrons to ETS
Anaerobic
To lactate
To ethanol

13. Glucose enters cells Overview of glycolysis in animal metabolism
Glycolysis, cont…
Overview of glycolysis in animal metabolism
Glucose in the blood
From breakdown of polysaccharides
Liver glycogen
Dietary sources
Gluconeogenesis
Synthesis from noncarbohydrate precursors
Glucose enters cells
Specific transporters

14. Enzymes of glycolysis in cytosol
Glycolosis, cont…
Enzymes of glycolysis in cytosol
Glucose converted into 2 3-carbon unites (pyruvate)
Free energy harvested to synthesis ATP from ADP and Pi
Pathway of chemically coupled phosphorylation reactions
10 reactions broken into 2 phases
Preparatory phase (energy investment)
Reactions 1 – 5
Payoff phase (energy recovery)
Reactions

15. Preparatory phase (energy investment)
Glycolosis, cont…
Preparatory phase (energy investment)
Hexose glucose is phosphorylated by ATP
C3-C4 bond broken
yields 2 triose phosphates (glyceraldehyde -3-phosphate)
Requires 2 ATP to “prime” glucose for cleavage

17. Payoff Phase (energy recovery) Each triose phosphate is oxidized
Glycolosis, cont…
Payoff Phase (energy recovery)
Each triose phosphate is oxidized
Energy is conserved
by reduction of NAD+
Phosphate is transferred to ADP  ATP
Net gain: 2 ATP
2 Glyceraldehyde-3-phosphate molecules are converted to 2 molecules of pyruvate
NadH must be reoxidized

19. ATP formation is coupled to glycolysis
Glycolosis, cont…
ATP formation is coupled to glycolysis
Glucose  pyruvate generates 2 ATP (net)
Involves coupled reactions
Makes glycolysis irreversible under intracellular conditions
Most energy remains in pyruvate
Glycolysis releases ~ 5%
Oxidation via TCA cycle releases the rest

20. Phosphorylated intermediates are important
Glycolosis, cont…
Phosphorylated intermediates are important
Each intermediate is phosphorylated
Phosphate has 3 functions:
Prevent diffusion of the intermediates out of the cell
Can donate Pi to ADP  ATP
Provide binding energy to increase specificity of enzymes

21. The Reactions of Glycolysis
10 enzymes
9 Intermediates
Cost (2 ATP)
Payment
4 ATP
2 NADH +H+
End products
Metabolic crossroads

22. Reaction 1 Intermediate formed: Glucose-6-phosphate (G6P)
Hexokinase: First ATP Utilization
Transfer of a phosphoryl group
From ATP
To glucose (at C-6)
Intermediate formed: Glucose-6-phosphate (G6P)
Enzyme: Hexokinase
Allosterically inhibited by product
REGULATION SITE (one of three)
Reaction is irreversible

24. Reaction 1, cont…
Kinase: enzymes that transfers phosphoryl groups between ATP and a metabolite
Name of metabolite acceptor is in prefix of the kinase name
E.g.:
glucokinase (in liver) is specific for glucose
Hexokinase: ubiquitous, relatively nonspecific for hexoses
D-glucose
D-mannose
D-fructose

25. Reaction 1, cont… Second substrate for kinases (including hexokinase)
Mg2+ -ATP complex
Mg2+ is essential
Uncomplexed ATP is a potent inhibitor of hexokinase
Mg2+ masks negative charge on phosphate oxygen atoms
Makes nucleophilic attack by C6-OH group on gamma-phosphorus atom more possible

26. Reaction 1, cont…

27. Substrate induced conformational changes in yeast hexokinase
Glucose (magenta) induces significant change … like jaws … this places ATP in close proximity to the C6-H2OH group and excludes water (which prevents ATP hydrolysis)

28. Reaction 1, cont… Begins glycolysis Is first of 2 priming reactions
Reaction is favorable under cellular conditions
Hydrolysis of ATP: liberates 30.5 kJ/mol
Phosphylation of glucose: costs 13.8kJ/mol
Delta G= kJ/mol

29. Reaction 1, cont… Importance of phosphorylating glucose
Keeps substrate in the cell
Glucose enters cell via specific transporters
The transporter does not bind to G6P
G6P is negatively charged, thus can not pass through plasma membrane
Rapid phosphorylation of glucose keeps intercellular concentrations of glucose low
Favors diffusion into cell
Regulatory control can be imposed only on reactions not at equilibrium
Large negative free energy change make this an important site for regulation

30. Reaction 1, cont… Glucokinase In liver
Carries out same reaction, but is glucose specific (high Km for glucose)
Not inhibited by the product
Important when blood glucose levels are high
Glucose to G6P to stored glycogen
Inducible by insulin
When blood glucose levels are low, liver uses hexokinase

31. Reaction 2 Intermediate formed: Fructose-6-phosphate (F6P)
Phosphoglucose Isomerase (PGI)
Conversion of G6P to Fructose-6-phosphate
Isomerization of an aldose to a ketose
Intermediate formed: Fructose-6-phosphate (F6P)
Enzyme: Phosphoglucose Isomerase
Reversible reaction

33. Reaction 2, cont… Common reaction: isomerization of a sugar
Requires ring of G6P to open
Isomerization
Ring of F6P closes
Prep for next reactions
R3: Phosphorylation at C-1
R4: cleavage between C-3 and C-4
PGI in humans
Requires Mg2+
Highly specific for G6P
Reaction is near equilibrium, easily reversible
Small delta G value

34. Reaction 3
Phosphofructokinase: second ATP utilization
Phosphorylation of F6P to Fructose-1,6-bisphosphate
bis not di: phosphates not together)
ATP donates a phosphate
Intermediate formed: Fructose-1,6-bisphosphate (FBP or F1,6P)
Enzyme: Phosphofructokinase (PFK-1)
REGULATION SITE (two of three)
Irreversible reaction

36. Reaction 3, cont… Allosteric regulation of PFK in many organisms
Similar to Hexokinase reaction
Nucleophilic attack by C1-OH of F6P on Mg2+ -ATP complex
PFK plays central role in control of glycolysis
Catalyzes one of the pathway’s rate-determining reactions
Allosteric regulation of PFK in many organisms

37. Reaction 4 Intermediates formed: GAP and DHAP Enzyme: aldolase
Cleavage of Fructose-1,6-bisphosphate
Forms two trioses
Glyceraldehyde-3-phosphate (GAP)
Dihydroxyacetone phosphate (DHAP)
Intermediates formed: GAP and DHAP
Enzyme: aldolase
Reversible reaction

39. Reaction 4, cont… A cleavage between C-3 and C-4
two molecules from one
Requires:
A carbonyl at C-2
A hydroxyl at C-4
Hence the “logic” at reaction 2
2 classes of aldolases
Class I: in animal tissues
Class II: in bacteria and fungi
Require a active-site metal, normally zinc Zn 2+

40. Reaction 5 Intermediate formed: Glyceraldehyde-3-phosphate
Triose phosphate isomerase
Interconversion of DHAP and GAP (triose phosphates)
Isomerization of aldose-ketose isomers
Intermediate formed: Glyceraldehyde-3-phosphate
Enzyme: Triose phosphate isomerase
Reversible reaction

43. Reaction 5, cont … Only glyceraldehyde-3-P can continue in glycolysis
Dihydroxyacetone-P is rapidly converted

45. Taking Stock so far Investment phase: Produces 2 triose phoshates
One glucose  2 glyceraldehyde-3-P
Costs 2 ATP
Now, need a little chemical “artistry” to convert low energy GAP to high energy compounds and synthesis ATP

46. Next … Payoff phase: Produces ATP One glucose  2 glyceraldehyde-3-P
Conversion to pyruvate  4 ATP
Also 2 reduced NADH

47. Reaction 6 Intermediate formed: 1,3-bisphosphoglycerate
Glyceraldehyde-3-phosphate Dehydrogenase: First “High-energy” Intermediate Formation
Oxidation of GAP by NAD+ and Pi
Intermediate formed: 1,3-bisphosphoglycerate
Enzyme: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
Reaction is reversible
Energy-conserving reaction

49. Reaction 6, cont …
Aldehyde is dehydrogenated to an acyl phosphate with a high standard free energy of hydrolysis (ΔG01 = kJ/mole)
NAD+ serves as hydrogen acceptor:
NAD+  NADH + H+

50. Reaction 7 Intermediate formed: 3-phosphoglycerate
Phosphoglycerate kinase: first ATP generation
Transfer of a phosphate to ATP
Yields ATP & 3-phosphoglycerate
Intermediate formed: 3-phosphoglycerate
Enzyme: PHOSPHOGLYCERATE KINASE
Energy-coupling reactions 6 & 7
A substrate-level phosphorylation

52. Enzyme: PHOSPHOGLYCERATE MUTASE (PGM)
Reaction 8
Conversion of 3 PG to 2-phosphoglycerate (2PG)
Intermediate formed:
Enzyme: PHOSPHOGLYCERATE MUTASE (PGM)
Reversible phosphate shift

54. Reaction 9 Enzyme: ENOLASE Intermediate formed: phosphoenol pyruvate
Dehydration to Phosphoenol Pyruvate (PEP)
Intermediate formed: phosphoenol pyruvate
Enzyme: ENOLASE
Energy-conserving reaction
Reversible reaction

56. Enzyme: Pyruvate kinase
Reaction 10
Pyruvate kinase: Second ATP generation
Transfer of a phosphate to  ATP
Product: pyruvate
Enzyme: Pyruvate kinase
Irreversible reaction
Substrate-level phosphorylation
“enol” spontaneously tautomerizes to “keto” form

59. Overall balance sheet:
Glycolosis, cont…
Overall balance sheet:
Anaerobic:
net gain of 2 ATP
Must “free” reduced NAD from reaction 6
In humans: lactic acid pathway
Aerobic:
NADH re-oxidized to NAD+ via respiratory chain in mitochondria
e- transfer provides energy for ATP synthesis
2.5 ATP/ reduced NAD
Therefore: 5 more ATPs if go aerobic

60. Anaerobic alternatives for pyruvate
Glycolosis, cont…
Anaerobic alternatives for pyruvate
Must oxidize NAD
Lactic acid pathway
Fermentation
Aerobic alternatives for pyruvate
Hydrogens from reduced NAD transported to ETS in mitochondria
Transporters in mitochondrial membrane

63. Dietary polysaccharides: Dietary Disaccharides:
Glycolosis, cont…
Dietary polysaccharides:
must by hydrolyzed to monosaccarides
Dietary Disaccharides:
Disaccharides cannot enter glycolytic pathway

65. Hexoses can enter glycolysis
Glycolosis, cont…
Hexoses can enter glycolysis
Hydrolytic enzyes are attached to epithelial cells in intestines
Monosaccharides  intestinal cells  blood  liver (phosphorylation)  glycolysis

67. III. REGULATION of CARBOHYDRATE CATABOLISM
Regulatory enzymes act as metabolic valves
Substrate-limited reactions are determined by [S]
Enzyme-limited reactions are RATE-LIMITING STEPS
Irreversible reactions
Exergonic
regulatory

68. Regulation of Carbohydrate Catabolism, cont…
Regulation of glucose metabolism differs in muscle & liver
Muscle: Object is ATP production
Enzyme: GLYCOGEN PHOSPHORYLASE
Enzyme is allosterically regulated
Skeletal muscle signalled to  ATP by EPINEPHRINE
Both enzyme & hormone influence ATP production

69. Regulation of Carbohydrate Catabolism, cont…
Liver: object is maintenance of blood glucose levels
Regulated by GLUCAGON & [blood glucose]
Enzyme: GLUCOSE-6-PHOSPHATE ↓
GLUCOSE-6-P + H2O  GLUCOSE + Pi

70. Regulation of Carbohydrate Catabolism, cont…
Other regulatory enzymes
Hexokinase: catalyzes entry of free glucose into gycolysis
Pyruvate kinase: catalyzes last step in glycolysis
Inhibited by ATP, excess fuel

71. Regulation of Carbohydrate Catabolism, cont…
Phosphofructokinase-1: commits cell to passage of glucose through glycolysis
Irreversible reaction
Allosterically inhibited by ↑ [ATP]
When ATP levels are sufficiently high, glycolysis is turned down
Inhibition relieved by allosteric action of ADP & AMP
Rate of glycolysis increases when ATP levels are low

72. Regulation of Carbohydrate Catabolism, cont…
Phosphofructokinase-1: links glycolysis and citric acid cycle (CAC)
Allosterically inhibited by citrate
An intermediate in CAC
When citrate accumulates, glycolysis slows down
Phosphofructokinase-1also regulated by beta-D-fructose-2,6-bisphosphate
Allosteric activator
Increases affinity of PFK for F6P

73. Regulation of Carbohydrate Catabolism, cont…
Futile Cycling: simultaneous production & consumption of glucose by the cell
Gluconeogenesis: conversion of pyruvate  glucose (opposite of glycolysis)
Uses some of the same enzymes as glucolysis

74. Regulation of Carbohydrate Catabolism, cont…
Both sets of reactions are substrate limited
Some glycolytic reactions are irreversible (3, catalyzed by regulatory enzymes)

75. Regulation of Carbohydrate Catabolism, cont…
These reactions are by-passed in gluconeogenesis by different enzymes
To prevent FUTILE CYCLING, enzymes limited reactions are subject to reciprocal allosteric control

76. IV. SECONDARY PATHWAYS of GLUCOSE OXIDATION
Pentose Phosphate pathway
Produces NADPH & ribose-5-phosphate
NADPH used in biosynthesis of fatty acids, steroids
Pentoses used in nucleic acid synthesis

77. Secondary Pathways of Glucose Oxidation, cont…
Transformation into Glucuromic Acid & Ascorbic Acid
D-glucuronate: used to convert non-polar toxins to polar derivatives
L-ascorbic acid: cannot be accomplished by humans