Metabolim of fructose and Galactose Fructosuria Hereditary Fructose intolerance Galactosira Lactose intolerance Monosaccharide mal-absorbtion

1. Glycolysis and Its Importance
Dr. Endriyas Kelta (DMD, MSc)
Assistant Professor, AAU

2. Glycolysis
– Also called Embden-Meyerhof Pathway
– Series of enzyme-catalyzed reaction pathway
• Glucose is oxidized into pyruvate
– Takes place in cytosol of all cells
– Major pathway for glucose utilization
– Unique pathway
• Functional both in the presence & absence of oxygen
– Universal central pathway of glucose catabolism
in all forms of life
• Differs by means of regulation & fate of pyruvate
– Free energy released is captured in the form of
ATP and NADH
2

3. Fates of Pyruvate 3

4. Cont…
• Glycolysis takes place in two phases:
– Preparatory Phase
• Consists of 1st five reaction steps
• One molecule of glucose is starting material
• Two molecules of Glyceraldehyde 3-phosphates
are end products
• Two molecules of ATP are invested
– Payoff phase
• Consists of 2nd five reaction steps
• Two molecules of Glyceraldehyde 3-phosphates
are starting materials
• Two molecules of pyruvates are end products
• Four molecules of ATP & 2 molecules of NADH +
H+ are conserved 4

5. Cont…
• Three types of chemical transformations are
noted:
– Degradation of the carbon skeleton of glucose to
pyruvate
– Phosphorylation of ADP to ATP by high-energy
phosphate compounds formed during glycolysis
– Transfer of a hydride ion to NAD+, forming
NADH
5

6. Cont…
• Glycolytic pathway involves 10 cytosolic enzymes
• Most glycolytic enzymes require Mg2+ for their
activity
– Phosphate groups of ADP, ATP, & glycolytic
intermediates form complexes with Mg2+
– Substrate binding sites of most glycolytic enzymes are
specific phosphate-Mg2+ complexes
6

7. Individual Reactions of Glycolysis
• Phosphorylation of Glucose:
– 1st priming reaction of glycolysis
– Essentially irreversible reaction
– Activation of glucose for subsequent reactions
– Glucose is phosphorylated at C-6 to yield
glucose 6-phosphate
– Glucose-6-phosphate:
• Product of glucose phosphorylation reaction
• Cross road molecule
– Glycolysis
– Glucuronic acid synthesis
– Glycogenesis
– Pentose phosphate pathway
7

8. Cont…
– ATP is phosphoryl group donor
– Two kinases are involved:
• Hexokinase
• Glucokinase
8

9. Hexokinase Vs Glucokinase
• Hexokinase:
– Founds in all cells
• Exceptions:
– Liver parencymal cells
– Pancreatic islet cells
– High affinity for glucose
• Functional even in low conc. of glucose
– Ensures the supply of glucose for the tissue
– Maintains high conc. of glucose inside cell
– Found in fetal liver
– Inhibited by its product glucose-6- phosphate
9

10. Cont…
• Glucokinase:
– Founds in liver parencymal & pancreatic islet
cells
• Never found in fetal liver
– Low affinity for glucose
• Functional in high conc. of glucose
– E.g., After a meal rich in carbohydrate
– Traps all available glucose regardless of glucose 6-
phosphate conc.
» Permits storage of glucose as glycogen or fatty
acids
– Its activity is enhanced by insulin
• E.g., Type-1 diabetic patients ( insulin deficiency) 10

11. Cont…
• Conversion of Glucose 6-Phosphate to
Fructose 6-Phosphate
– Reversible isomerization of an aldose sugar to a
ketose sugar
– Catalyzed by phosphohexose (glucose) isomerase
11

12. Cont…
• Phosphorylation of Fructose 6-Phosphate
to Fructose 1,6- Bisphosphate
– 2nd priming reactions of glycolysis
• Fructose 6-phosphate is phosphorylated at C-1 to yield
Fructose 1, 6-Bis-phosphate
• Catalyzed by phosphofructokinase-1 (Called so, to
distinguish it from Phosphofructokinase-2)
12

13. Cont…
• PFK-1 reaction is essentially irreversible
• PFK-1 is a key regulatory enzyme in glycolysis
– Fructose 1,6-bisphosphate is not a cross road
molecule
• Cleavage of Fructose-1,6-Bisphosphate
– This reaction step is readily reversible
– Yields two triose phosphates:
• Aldotriose phosphate: glyceraldehyde-3-phospate
(GA3P)
• Ketotriose phosphate: dihydroxyacetone phosphate
(DHAP)
– Catalyzed by Fructose-1 ,6-bisphosphate
aldolase (aldolase)
• Doesn’t require Mg2+ for its activation 13

14. 14

15. Cont…
• Interconversion of Triose Phosphates
• Reversible isomerization of a ketose sugar into
an aldose sugar
• DHAP rapidly and reversibly converted to GA3P
• Catalyzed by triose phosphate isomerase
• Doesn’t require Mg2+ for its activation
15

16. Cont…
• Oxidation of Glyceraldehyde-3-Phosphate
to 1,3-Bisphospho-glycerate
– 1st reaction step in payoff phase of glycolysis
– GA3P is oxidized into 1,3-bisphosphoglycerate
• Lost 2e- & 2H+
– Catalyzed by NAD+ & inorganic phosphate(pi)
dependant, GA3P Dehydrogenase
• Doesn’t require Mg2+ for its activation
– Coenzyme NAD+ is reduced into NADH +H+
• Gain 2e- + H+ (hydride ion (H-)
• One proton exist in solution as H+
16

17. 17

18. Cont…
• Substrate Level Phosphorylation Reaction
– High-energy phosphoryl group is transferred
from 1 ,3-bisphosphoglycerate to ADP
• ATP & 3-phosphoglycerate are formed
– Catalyzed by phosphoglycerate kinase
18

19. Cont…
• Clinical comments:
– Arsenate uncouples the formation of ATP at this
stage
• Competes with phosphate in GA3P dehydrogenase reaction
– 1-Arseno-3-phosphoglycerate is formed
» Unstable & spontaneously hydrolyzed into 3-
phosphoglycerate & heat without generating ATP
– In erythrocytes, formation of ATP is bypassed at
this stage
• When cellular need for ATP is minimal
– 1,3-bisphosphoglycerate converted into 2,3-BPG by
bisphosphoglycerate mutase
– 2,3-BPG hydrolyzed into 3-phosphoglycerate by 2,3-
bisphosphoglycerate phosphatase without generating
ATP 19

20. 20

21. Cont…
• Conversion of 3-Phosphoglycerate to 2-
Phosphoglycerate
– Reversible shift of the phosphoryl group b/n C-2
& C-3 of Phosphoglycerate
– Catalyzed by phosphoglycerate mutase
21

22. Cont…
• Dehydration of 2-Phosphoglycerate to
Phosphoenolpyruvate (PEP)
– Reversible removal of a molecule of water from
2-phosphoglycerate to form PEP
– Catalyzed by Mg+2 or Mn+2 dependent enzyme,
enolase
22

23. Cont…
• Clinical comments:
– Fluoride ion
• Inhibits enolase activity
• Required to prevent glycolysis in blood sample
prior to lab. estimation of glucose
• Substrate Level Phosphorylation Reaction
– High-energy phosphoryl group is transferred
from PEP to ADP
• ATP & pyruvate are formed
– Catalyzed by pyruvate kinase
– Requires K+ & either Mg2+ or Mn2+
23

24. 24

25. Cont…
• Pyruvate kinase reaction is essentially
irreversible
– Pyruvate appears in equilibrium b/n enol form &
keto form (reversible reaction)
– Enol form rearranges itself spontaneously
(tautemerization)keto form
• Favors formation of ketoform (pulls equilbm. towards
right)
25

26. Regulation of Glycolysis
• Major sites of regulation of glycolysis:
– Physiologically irreversible reaction steps
catalyzed by
• Hexokinase & Glucokinase
• PFK-1
• Pyruvate kinase
(1) Regulation at Hexokinase Level:
– Inhibited by high conc. of glucose 6-phosphate
(2) Regulation at Glucokinase Level:
– Indirectly:
• Inhibited by high conc. of fructose 6-phosphate
• Stimulated by high conc. of glucose 26

27. Cont…
• In high conc. fructose 6-phosphate
– Glucokinase
• Translocated into hepatocyte nucleus
• Reversibly binds with Glucokinase Regulatory protein
(GKRP)
• Its activity is inhibited
• In high conc. of blood & hepatocyte cytosolic
glucose:
– Glucokinase
• Released from GKRP & Re-enters cytosol
• Its activity is stimulated
27

28. Regulation of glycolysis at GK level 28

29. Cont…
(3) Regulation at PFK-1 Level:
– (a) Regulated by fructose 2,6-bisphosphate
• Fructose 2,6-bisphosphate
– Formed from fructose 6-phosphate by PFK-2
activity
– Most potent activator of PFK-1 (Even when ATP
levels are high)
• PFK-2 is a bifunctional protein
– Kinase activity Fructose 2,6-bisphosphate
» Dephosphorylation  Active kinase domain
» Phosphorylation  Inactive kinase domain
– Phosphatase activity Fructose 6-phosphate
29

30. Cont…
• Decreased glucagon/insulin ratios (during fed state):
– Increase in fructose 2,6-bisphosphate
– Increase in rate of glycolysis in the liver
• Elevated glucagon/insulin (during fasting)
– Decrease in fructose 2,6-bisphosphate
– Decrease in the overall rate of glycolysis
– (b) Regulated by energy rich & poor signals
within the cell:
• Energy rich signals (ATP & citrate) inhibit PFK-1
Activity
• Energy poor signals (AMP & ADP) stimulate PFK-1
Activity
30

31. Regulation of PFK-1 31

32. Effect of elevated glucagon/insulin ratio in
overall rate of glycolysis 32

33. Effect of decreased glucagon/insulin ratio in
overall rate of glycolysis 33

34. Cont…
(4) Regulation at pyruvate kinase Level:
– Feed-forward regulation:
• Activated by fructose 1,6-bisphosphate
– Regulation by covalent modification:
• Phosphorylation  Inactivates PK
• Dephosphorylation  Activates PK
• Elevated glucagon/insulin (during fasting) 
increased cAMP Causes phosphorylation of PK 
Inhibition of overall rate of glycolysis
• Decreased glucagon/insulin ratios (during fed
state)  decreased cAMP  causes
dephosphorylation of PK (phosphoprotein
phosphatase)  stimulates overall rate of
glycolysis 34

35. Regulation of PK by covalent modification
35

36. Regulation of Hexokinase, PFK-1 & pyruvate
kinase
36

37. Hormonal regulation of Glycolysis 37

38. Overall Energy Balance Sheet of glycolysis
• Overall Energy Balance
– Accounts for the fate of:
• Carbon skeleton of glucose
• Phosphoryl groups
• Electrons in redox reactions
– Under two conditions:
• Aerobic glycolysis
• Anaerobic glycolysis
38

39. Overall Energy Balance of Aerobic glycolysis
– Over all reaction equation for aerobic glycolysis
• Glucose + 2ATP + 4ADP + 2Pi + 2NAD+  2Pyruvate +
4ATP + 2ADP + 2NADH +2H+ +2H2O
– When common terms are cancelled out:
• Glucose + 2ADP +2Pi +2NAD+ 2pyruvate + 2NADH
+2H+ + 2ATP +2H2O
– Fate of the carbon skeleton of glucose
• One molecule of glucose  Two molecules of pyruvate
– Fate of phosphoryl groups
• Two molecules of ADP and Pi  Two molecules of ATP
– Fate of Electrons
• Four electrons transferred into two molecules of
NAD+  Two molecules of NADH
39

40. Cont…
• In animal tissue, supply of NAD+ is very
limited
– Glycolytic pathway is going to stop
• Cytosolic NADH+ H+ should be re-oxidized
– For re-oxidation (under aerobic conditions):
» Cytosolic NADH + H+ should be transferred into
mitochondrial matrix
• Inner mitochondrial membrane is, however,
impermeable for cytosolic NADH + H+
– Two special means of transport are needed:
» Glycerol 3-Phosphate Shuttle system (GPS)
» Malate–Aspartate Shuttle system (MAS)
40

41. Glycerol 3-Phosphate Shuttle system (GPS)
41

42. Malate–Aspartate Shuttle system (MAS)
42

43. Overall Energy Balance of Anaerobic glycolysis:
– Anaerobic glycolysis takes place in:
• Exercising skeletal muscles
• Erythrocytes
• Lactic acid fermenting bacteria
– Over all reaction equation is:
• Glucose + 2ATP + 4ADP +2Pi + 2NAD+ + 2NADH + 2H+
2lactate +4ATP + 2ADP + 2NAD+ + 2NADH + 2H+ +
2H2O
– When common terms are cancelled out:
• Glucose + 2ADP +2Pi 2lactate + 2ATP + 2H2O
43

44. – Fate of the carbon skeleton of glucose
• One molecule of glucose  Two molecules of
Lactate
– Fate of phosphoryl groups
• Two molecules of ADP and Pi  Two molecules of
ATP
– Fate of Electrons (no net gain of electrons)
• Four electrons transferred into two molecules of
NAD+  Two molecules of NADH (reutilized)
• Here,
– Two molecules of NADH are (reutilized) by
Lactate dehydrogenase reaction
• To regenerate NAD+ for continuation of glycolysis
44

45. 45

46. Cont…
– Anaerobic glycolysis also takes place in:
• Alcohol fermenting microorganisms, E.g., yeasts
46

47. Cont…
– Over all reaction equation for alcohol
fermentation is:
• Glucose + 2ATP + 4ADP + 2Pi + 2NAD+ + 2NADH + 2H+
2Ethanol + 4ATP+ 2ADP + 2NAD+ + 2NADH + 2H+ +
2H2O+ 2CO2
– When common terms are cancelled out:
• Glucose +2Pi + 2ADP===2Ethanol + 2ATP + 2H2O +
2CO2
– Fate of the carbon skeleton of glucose
• One molecule of glucose  Two molecules of Lactate
– Fate of phosphoryl groups
• Two molecules of ADP and Pi  Two molecules of ATP
47

48. Cont…
– Fate of Electrons (no net gain of electrons)
• Four electrons transferred into two molecules of
NAD+  Two molecules of NADH (reutilized)
• Here,
– Two molecules of NADH + H+ are (reutilized) by
Alcohol dehydrogenase reaction
• To regenerate NAD+ for continuation of glycolysis
48

49. Thank you!
49