1. Biochemistry Department
Glycolysis
By Dr. Wael Elayat

2. By the end of this lecture You will be able to:
Intended Learning Outcomes
By the end of this lecture You will be able to:
Define glycolysis.
Understand all the steps involved in glycolysis.
Be able to calculate the energy yield.
Know the other substrates for glycolysis.
Understand the differences that exist in glycolysis in certain tissues.
Explain how glycolysis is controlled
Enumerate different functions of glycolysis
Explain manifestations of pyruvate kinase deficiency

3. Glycolysis

4. (Embden- Meyerhof cycle)
Glycolysis
(Embden- Meyerhof cycle)
Definition: Oxidation of glucose for energy production. Site? (Compartmentation)” Cytoplasm of all cells and tissues

5. When? It is an emergency cycle occurring All the time depending on:
1- Availability of substrate
2- Energy state of the cell
So it could be inhibited In:
1- The high energy state i.e. ( ATP ) .
2-Presence of alternative source of energy (as indicated by high level of citrate)
3- Absence of substrate (Starvation)

6. Out comes??? Pyruvate in cells with mitochondria &
of adequate supply of oxygen.
Lactate
in cells with No mitochondria “RBCs” &
Inadequate oxygen supply “Exercising muscles” .

7. Tissues Dependent On Glycolysis
Brain:
Dependent on glucose as primary source of fuel
(rapid source of ATP)
RBC:
Dependent on glucose for energy production.
Lack mitochondria
(No FA oxidation, No TCA, No ETC)
(So It depends on glycolysis)
Exercising Muscles (sever exrcise):
Anaerobic condition (No O2)…..(No ETC)

8. (No FA oxidation, No TCA, No ETC) (So thy depend on glycolysis)
Other tissues: (anaerobic tissue)
Cornea, lens, retina, testes, white muscle fiber, renal medulla and white blood cells
Dependent on glucose (as RBCs) for energy production.
Lack mitochondria
(No FA oxidation, No TCA, No ETC)
(So thy depend on glycolysis)

9. Reactions of glycolysis
Pass through 2 phases
Preparatory Phase (1st 5 steps).
Payoff Phase (Last 5 steps).
Or 3 stags
1- Priming stage (1st 3 steps).
2- Splitting stage (4th step).
3- Oxidative phosphorylation stage (Rest of steps)

10. Preparatory Phase (Energy Consuming phase) It involves 5 steps

11. Phosphorylation of glucose

12. Hexokinase or glucokinase (hepatic isomer of hexokinase)
CH2- OH
Glucose
CH2O- P
Glucose-6-P
Hexokinase
Mg++
ATP
ADP

13. Significance of phosphorylation of glucose?

14. 1-To Trap the Glucose inside the cell
-Ve Phosphate Group &–Ve Charge Of Cell Membrane
→ Repulsion !!
2- Maintain glucose concentration always low inside the cell

15. Phospho hexo Isomerase
Hexose-Isomerase
Phospho hexo Isomerase

16. Phospho Fructo kinase PFK- I
Mg++
NAD
NADH+H+

17. Aldolase A
Aldolase A
Remember : Aldolase B !!

18. F-6-P =O ↔ P P P Isomerase Glucose G-6-P F1-6-b P CH2- OH CH2O - P
ATP
Kinase
CH2O - P
G-6-P
Isomerase
F-6-P
CH2O - P
ATP
Kinase
F1-6-b P
CH2O - P
O - P ↔ P =O
DHAP
G3 -P P P
Glyceraldehyde 3-P
Isomerase

19. Payoff Phase (Energy Producing phase) It involves 5 steps

20. G3P Dehydrogenase

21. Phospho glycerate kinase

22. Phospho glycerate Mutase (Late)

23. Enolase

24. Pyruvate Kinase

25. Lactate dehydrogenase (anaerobic glycolysis)

26. Glycolysis

27. Biological Importance Of Glycolysis

28. Energy production
Anaerobic glycolysis
Gain 4 ATP by substrate level phosphorylation and loss 2 ATP for activation. So net gain is 2 ATP
Aerobic glycolysis
Gain 4 ATP by substrate level and 6 ATP by oxidative phosphorylation (from oxidation of 2 NADH+H in electron transport chain in mitochondria), and loss 2 ATP for activation. So net gain is 6 ATP

29. Mechanism of energy production
1- Substrate level phosphorylation Production of ATP directly from the reaction 2- Oxidative phosphorylation Production of ATP from oxidation of reduced equivalent in electron transport chain (ETC).

30. 2- Synthesis of 2,3 BPG
Some of the 1, 3 BPG is converted to (2,3-BPG) by the action of ↔ bisphosphoglycerate mutase . 2,3-BPG, is found in only trace amounts in most cells & in high concentration in RBCs.
2,3-BPG is hydrolyzed by Phosphatase Enzyme
→ 3-PG, which is also an intermediate in glycolysis.

31. Glycolysis in Red cells (RL-cycle Or Shunt) (Rapaport - Lubering cycle )

33. Importance Of 2,3 BPG in RBCs
It ↓ the affinity of hemoglobin for O2
→ facilitate the release of O2 to the tissues
(protect against hypoxia).

34. 3- Synthetic function
glycolysis can be used for synthesis of other compounds, such as :
DHAP → Glycerol 3 P (for TAG synthesis in adipose tissue)
Non-essential amino acid alanine (as serine from PSP and alanine from pyruvate).

35. Inhibitors of glycolysis (Arsenate)
Arsenate (pentavalent) ↓ net ATP and NADH production in glycolysis, without inhibiting the pathway itself. By competing with Pi

36. Phosphoglycerate kinase
G3P Dehydrogenase
NAD
NADH + H Pi
G3P D
Mg++
Phosphoglycerate kinase

37. G3P Dehydrogenase
NAD
NADH + H AS
G3P Dehydrogenase Pi

38. (Arsenate)
Act as a substrate for G3P D forming an unstable compound that spontaneously hydrolyzes forming 3-PG without ATP !!!!!!!!!!!! by passing 1 ,3-BPG synthesis & dephosphorylation this will deprive the cell of an ATP obtained by G3PD.

39. Inhibitors of glycolysis (Fluoride)
Fluride in the tooth paste inhibit the activity of Enolase Enzyme So it can inhibit glycolysis in bacteria So tooth past can prevent the teeth decay!!

40. Regulation Of Glycolysis

41. All the 3 Irreversible Enzymes
Regulatory enzymes
All the 3 Irreversible Enzymes
Glucokinase (Hexokinase)
PFK 1
Pyruvate kinase

42. Long Term Regulation
Slow hormonal influences on the amount of enzyme protein synthesized ……In the well fed state glucose is high →  insulin &  Glucagon glucose utilization is enhanced & ↑in the amount of Glucokinase (not hexokinase). (↑gene transcription) in liver

43. Glucokinase (Long term)

44. Glucokinase is induced in well fed state this allows control of glucose level especially after meals since it has a high Km.

45. Glucokinase also controlled indirectly by fructose 6 phosphate and glucose.
1-In presence of fructose 6 phosphate, glucokinase trans located to the neculus and bind to glucokinase regulatory protein and thus the enzyme will be inactive.
2- In presence of high blood glucose, glucoknase reenter cytoplasm and become active.

46. Hexokinase
(Allosteric)

47. even when its concentration is low
a constitutive Enzyme (Not induced)
Deal with glucose
even when its concentration is low
 V. Important for brain (Low Km)!!
Allosterically stimulaed by  ATP/ADP
Allosterically Inhibited by  G6-P

48. Student activity
Compare and contrast between hexokionase and glucokinase?

49. PFKI
(Allosteric)

50. Allosterically activated by : 1- AMP 2- Fructose 2,6 bisphosphate (potent activator) Allosterically Inhibited by: 1- ATP 2- High level of citrate 3- Low pH

51. Source of F 2,6 bisphosphate
Fructose 2,6 bisphosphate is a product of action of a hepatic enzyme called PFK-2 on fructose 6 phosphate.

52. Is a bifunctional enzyme
PFK2
Is a bifunctional enzyme
It has both kinase and phosphatase activity,
The balance between them determines the level of fructose 2,6 bisphosphate
and hence control Rate of glycolysis in cells.

53. PFKII
(Covalent)

54. (Bifunctional enzyme)
The activity of this
(Bifunctional enzyme)
is controlled by covalent modification (phosphorylation-dephosphorylation)
in response to insulin/glucagons ratio. .

57. (Allosteric & Covalent)PK
(Allosteric & Covalent)

59. Allosteric Regulation Of PK
Allosteric stimulation of PK
In liver by (AMP and F 1,6-bp )
Allosteric Inhibition of PK
In liver by ATP

60. In Fasting it is inactivated by
Pyruvate kinase
In Fasting it is inactivated by
Kinase
(Phosphorylation)
→ inactivation in the liver.

61. In well fed state is Activated by
Pyruvate kinase
In well fed state is Activated by
Phosphatase
(Dephosphorylation)
→ activation in the liver.

62. Hormonal regulation of glycolysis
1- Short term regulation:
Hormone can momentary control rate of glycolysis (in time of minutes and hours) by covalent modification.
In well feed state where insulin/glucagon ratio is high, this favor dephosphorylation and activation of pyruvate kinse and PFK-2 kinase activity (with increased production of F2,6 BP and increased rate of glycolysis.
In fasting state: typically the reverse.

63. 2- Long term regulation
Hormones can also control rate of glycolysis over long period of time (hours or days), by inducing (increase amount) of enzymes of glycolysis.
In well feed state, key regulatory enzymes of glycolysis (glucokinase, PFK-1 and PK) will be induced under influence of high insulin level and this will increase rate of glycolysis.

64. Inborn error of glycolysis 1- Pyruvate kinase deficiency
Nature: Autosomal recessive disorder Clinical picture: The patient will be presented by symptoms of hemolytic anemia Anemia + jaundice

65. Cause of hemolysis of RBCs in PK deficiency
Pyruvate kinase deficiency → Impaired glycolysis in RBCs with no ATP production →→ Loss of Na/K pump activity →→ Dehydration of RBCs →→ Hemolysis and heme breakdown →→ High levels of bilirubin (mainly indirect)

66. N.B.: 1- The symptoms of anemia in PK deficiency will be ameliorated by the high level of accumulated 2,3 BPG which increase delivery of oxygen to the cells. 2- Explain why the liver has not been affected by this enzyme deficiency.

67. 1- Fate of end product of glycolysis
1- Fate of end product of anaerobic glycolysis
A- Fate of NADH+H+:
- As the cell has a limited amount of NAD, so NADH+H+ must be reoxdized to resynthesize NAD.
In absence of mitochondria and/or oxygen NADH+H+ is reoxdized by converting pyruvate to lactate

68. B- Fate of lactate
Diffuse out of anaerobic tissue cowith H+ to blood, then pass to liver where it will converted to glucose and go back to muscle. This cycle of lactate to glucose and then to lactate is known as Cori cycle and will be discussed in detail with gluconeogenesis.

69. Cori Cycle
Lactate
Glucose

70. 2- Fate of end products of aerobic glycolysis:
A- Fate of NADH+H+:
- As the cell has a limited amount of NAD, so NADH+H+ must be reoxdized to resynthesize NAD.
Reoxidation of NAD occur in mitochondria in ETC.
But the mitochondrial membrane impermeable to NADH+H+
NADH+H+ enter to mitochondria through one of shuttles across the mitochondrial membrane (Glycerol phosphate shuttle OR malate aspartate shuttle which will be discussed latter)

71. B- Fate of pyruvate
Converted to acetyl CoA by pyruvate dehydrogenase complex enzyme to be completely oxdized to CO2, H2O with production of more energy in TCA cycle.

72. Summary Of Important Points
Glycolysis is either :
Aerobic glycolysis, in which pyruvate is the end product (need O2 & mitochondria),
Anaerobic glycolysis, in which lactic acid is the end product. ( does not need O2 or mitochondria).
Glucose is transported across membranes by one of at least 14 glucose transporter iso-forms (GLUTs).
The conversion of glucose to pyruvate (glycolysis) occurs in 2 stages: Energy consuming phase in which phosphorylated intermediates are synthesized at the expense of ATP, and Energy producing phase, in which ATP is produced.
The 1st enzyme used with glucose is either Hexokinase (found in most tissues) or Glucokinase (a hexokinase found in liver cells and the β cells of the pancreas).

73. Questions to assess the ILOs
Choose the best answer:
1- Hexokinae enzyme:
Is the hepatic isoenzyme of glucokinase enzyme
Utilize Pi for trapping of glucose
Need Ca2+ as cofactor
Convert glucose to glucose 6 P
Non of the above

74. 2- Insulin can increase rate of glycolysis by:
Induction of hexokinase enzyme
Covalent activation of PFK-1
Indirect allosteric activation of PFK-1
Decreasing amount of fructose 2,6BPG
Activation of PFK-2 kinase part

75. 3- Glycolysis in RBCs:
Is an aerobic type of glycolysis
End by pyruvate
Can supply RBCs with net 4 molecules of ATP
Can be controlled by induction of glucokinase
End by lactate

76. Questions to assess the ILOs
Answer these questions PLZ…
Define: site, input, output & irreversible enzymes of glycolysis
Compare between Hexokinase & glucokinase enzymes
Differentiate between aerobic & aerobic glycolysis as regard the: O2 supply, net ATP consumption & production, one example for both
Define tissues that are dependent on anaerobic glycolysis & why ?!!

77. Explain the mechanism of action of toothpaste !
List the name of the 2 enzymes of glycolysis that are vital for RBCS as regard ATP production.
Explain the mechanism (enzymes used & substrates) of production of 2,3 BPG & its importance?
Explain the mechanism of action of toothpaste !
Explain the mechanism of action of Arsenic poisoning.
explain how lactic acid can be used by our body .
List 4 points importance of glycolysis (only)
Explain the regulatory mechanisms used in glycolysis as regard: types & enzymes regulated .
Dr. Amal Mansour

78. Thank you
Dr. Wael Elayat