glycolysis and TCA

1. By
Dr KHALED SALEH ALGARIRI
3 October 2018

2. INTRODUCTION
Glycolysis is the stepwise degradation of glucose (and
other simple sugars).
Carried out in the cytosol of cells, it is unique, in that it
can function either aerobically or anaerobically,
depending on the availability of oxygen and the electron
transport chain.
Glycolysis derived from Greek words.
Glykys= sweet ,Lysis= Splitting

3. Glycolysis consists of two phases-
• In the first, a series of five reactions, glucose is broken down
to two molecules of glyceraldehyde-3- phosphate.
• In the second phase, five subsequent reactions convert these
two molecules of glyceraldehyde-3- phosphate into two
molecules of pyruvate.
• Phase 1 consumes two molecules of ATP.
• The later stages of glycolysis result in the production
of four molecules of ATP.
• The net is 4 – 2 = 2 molecules of ATP produced per
molecule of glucose.

4. The First Phase of Glycolysis
 Reaction 1: Phosphorylation of Glucose by Hexokinase or
Glucokinase —
The First Priming Reaction
 • Glucose enters glycolysis by phosphorylation to glucose 6-
phosphate, catalyzed by hexokinase, using ATP as the
phosphate donor.
 • Under physiologic conditions, the phosphorylation of glucose
to glucose 6- phosphate can be regarded as irreversible

5. Significance of first priming reaction
Phosphorylation keeps the substrate in the cell. Glucose
is a neutral molecule and could diffuse across the cell
membrane, but phosphorylation confers a negative
charge on glucose, and the plasma membrane is
essentially impermeable to glucose-6-phosphate
• Rapid conversion of glucose to glucose-6- phosphate
keeps the intracellular concentration of glucose low,
favoring diffusion of glucose into the cell.

6. Significance of first priming reaction
 Phosphorylation of glucose to glucose-6-phosphate by ATP
creates a charged molecule that cannot easily cross the plasma
membrane.

7. First priming reaction
In most animal, plant, and microbial cells, the
enzyme that phosphorylates glucose is hexokinase.
• Magnesium ion (Mg2+) is required for this
reaction
• Hexokinase can phosphorylate a variety of hexose
sugars, including glucose, mannose, and fructose.
• Hexokinase reacts strongly with glucose, while its
affinity for fructose and galactose is relatively low.

8. Glucokinase occurs in cells in the liver, pancreas, gut,
and brain of humans and most other vertebrates.
 In each of these organs it plays an important role in
the regulation of carbohydrate metabolism by acting
as a glucose sensor, triggering shifts in metabolism or
cell function in response to rising or falling levels of
glucose, such as occur after a meal or when fasting.
• Mutations of the gene for this enzyme can cause
unusual forms of diabetes or hypoglycemia

9. Hexokinase versus Glucokinase

10. In the liver, the action of Glucokinase is opposed by
the action of glucose-6-phosphatase.
The balance between glucokinase and glucose-6-
phosphatase slides back and forth, increasing uptake
to the liver and phosphorylation when the level of
blood glucose is high, and releasing glucose from G-
6-P when blood glucose falls.
The function of glucokinase in the liver is to remove
glucose from the blood following a meal,
providing glucose 6-phosphate in excess of
requirements for glycolysis, which is used for
glycogen synthesis and lipogenesis.

11. Fate of Glucose-6-P
Glucose 6-phosphate is an important compound at
the junction of several metabolic pathways:
 Glycolysis
 Gluconeogenesis
 Pentose phosphate pathway,
 Glycogenesis
 Glycogenolysis

13. Energy yield per molecule of
Glucose oxidized through Glycolysis
Under anaerobic conditions Electron transport chain does not operate so
the ATP is only formed by substrate level phosphorylation. Hence the
total energy yield through glycolysis in the absence of oxygen is only 2
ATP per Mol of Glucose.

14. Regulation of Glycolysis
Flux through a metabolic pathway can be regulated
in several ways:
1. Availability of substrate
2. Concentration of enzymes responsible for rate-limiting steps
3. Allosteric regulation of enzymes
4. Covalent modification of enzymes (e.g. phosphorylation)

15. Regulation of Glycolysis (contd.)
 Enzymes that catalyze 3 irreversible steps in glycolytic pathways
are potential sites for regulatory control.
 • The enzymes responsible for catalyzing these three steps,
hexokinase (or glucokinase) for step 1, phosphofructo
kinase for step 3, and pyruvate kinase for step 10, are the
primary steps for allosteric enzyme regulation.
 • Availability of substrate (in this case, glucose), is another
general point for regulation.

16. Regulation of Glycolysis (contd.)
 The concentration of these three enzymes in the cell is regulated
by hormones that affect their rates of transcription.
 • Insulin upregulates the transcription of Glucokinase,
phosphofructo kinase, and pyruvate kinase, while glucagon
down regulates their transcription.
 • These effects take place over a period of hours to days, and
generally reflect whether a person is well-fed or starving

17. Regulation of Glycolysis (contd.)
1) Regulation at the level of Hexokinase and
Glucokinase
• The Hexokinase enzyme is allosterically inhibited by the product,
glucose-6-phosphate.
• Glucokinase is highly specific for D-glucose, has a much higher
Km for glucose (approximately 10.0mM ), and is not product-
inhibited.
• With such a high Km for glucose, Glucokinase becomes
important metabolically only when liver glucose levels are
high.
• Glucokinase is an inducible enzyme—the amount present in the
liver is controlled by insulin.

18. Regulation of Glycolysis (contd.)
2) Regulation of Phospho fructokinase
a) Role of ATP-
ATP is an allosteric inhibitor of this enzyme.
• In the presence of high ATP concentrations, the Km
for fructose-6-phosphate is increased, glycolysis thus
“turns off.
• AMP reverses the inhibitory action of ATP, and so the
activity of the enzyme increases when the ATP/AMP
ratio is lowered. In other words,glycolysis is stimulated
as the energy charge falls.

19. Regulation of Glycolysis (contd.)
2) Regulation of Phospho fructokinase
b) Role of Citrate
 Phosphofructokinase is inhibited by citrate, an early
intermediate in the citric acid cycle.
 A high level of citrate means that biosynthetic
precursors are abundant and additional glucose should
not be degraded for this purpose.
 Citrate inhibits phosphofructokinase by enhancing the
inhibitory effect of ATP

20. Regulation of Glycolysis (contd.)
2) Regulation of Phospho fructokinase
c) Role of Fr 2,6 bisphosphate
 Phosphofructokinase is also regulated by Dfructose-2,6-
bisphosphate, a potent allosteric activator that increases
the affinity of phosphofructokinase for the substrate
fructose-6- phosphate
Fructose-2,6-bisphosphate increases the net flow of
glucose through glycolysis by stimulating
phosphofructokinase and, by inhibiting fructose-1,6-
bisphosphatase, the enzyme that catalyzes this reaction
in the opposite direction.

21. Regulation of Glycolysis (contd.)
Why is phosphofructokinase rather than hexokinase the
pacemaker of glycolysis?
 Glucose 6-phosphate is not solely a glycolytic intermediate.
Glucose 6-phosphate can also be converted into glycogen or it
can be oxidized by the pentose phosphate pathway to form
NADPH.
 The first irreversible reaction unique to the glycolytic pathway,
the committed step, is the phosphorylation of fructose 6-
phosphate to fructose 1,6-bisphosphate.
 Thus, it is highly appropriate for phosphofructokinase to
 be the primary control site in glycolysis.

22. Regulation of Glycolysis (contd.)
3) Regulation of pyruvate Kinase
It is activated by AMP and fructose-1,6-bisphosphate and
inhibited by ATP, acetyl-CoA, and alanine.
• Liver pyruvate kinase is regulated by covalent modification.
• Hormones such as glucagon activate a cAMP dependent
protein kinase, which transfers a phosphoryl group from ATP
to the enzyme.
.

23. Regulation of Glycolysis (contd.)
 This hormone-triggered phosphorylation, prevents the
liver from consuming glucose when it is more urgently
needed by brain and muscles.

24. Inhibitors of Glycolysis
Inhibitors of Glycolysis
a) Arsenate and Iodoacetate- Inhibitors of Glyceraldehyde-3-
P dehydrogenase
b) Bromo hydroxy acetone phosphate- Inhibitor of dihydroxy
acetone phosphate
c) Fluoride- Inhibitor of Enolase
d) Oxamate- Inhibitor of Lactate dehydrogenase

25. Significance of glycolysis other
than energy production
Glucose-6-P is a common intermediate for a number of
pathways and is used depending on the need of the cell, like
glycogen synthesis, Uronic acid pathway, HMP pathway etc.
• Fructose-6-P is used for the synthesis of Glucosamines.
• Triose like glyceraldehyde-3-P and other glycolytic
intermediates can be used in the HMP pathway for the
production of pentoses.
• Dihydroxy Acetone –phosphate can be used for the synthesis
of Glycerol -3-P , which is used for the synthesis of Triglycerides
or phospholipids

26. Significance of glycolysis other
than energy production (contd)
 2,3 BPG is an important compound produced pathway in
erythrocytes in the glycolytic pathway for unloading of O2 to the
peripheral tissues.
 The sugars like Fructose, Galactose. Mannose and even Glycerol
can be oxidized in glycolysis.
 Out of the total 10 reactions of Glycolysis, 7 reactions are
reversible and are used for the synthesis of Glucose by the process
of Gluconeogenesis.
 Pyruvate the end product of glycolysis provides precursor for the
TCA cycle and for the synthesis of other compounds

27. WHAT HAPPENS TO PYRUVATE AFTER
IT IS MADE FROM GLYCOLYSIS.
In the presence of oxygen (aerobic condition) pyruvate is
converted to acetyl-CoA by the enzyme pyruvate dehydrogenase
which enters the TCA or Kerb cycle where large (most) of ATP
molecules is generated.
In the absence of oxygen (anaerobic conditions) pyruvate
undergoes fermentation either lactic acid fermentation or alcohol
fermentation. In this fermentation reaction NO ATP molecules is
generated, however reduced NAD+ is generated from
fermentation. The NAD+ regenerated is used in the glycolysis
process to make ATP. Therefore these cells only get energy (2
ATP) from glycolysis and not from the TCA cycle. Example of
such cell are red blood cells.

29. Citric acid cycle/ TCA/ krebs cycle
Definition
The citric acid cycle is the central metabolic hub of
the cell.
It is the final common pathway for the oxidation of
fuel molecule such as amino acids, fatty acids, and
carbohydrates .
 The reactions of the citric acid cycle take place inside
mitochondria, in contrast with those of glycolysis, which take
place in the cytosol.

30. Overview of the Citric Acid Cycle
The citric acid cycle (Krebs cycle, tricarboxylic acid
cycle) includes a series of oxidation reduction reactions
in mitochondria that result in the oxidation of an acetyl
group to two molecules of carbon dioxide and reduce the
coenzymes that are reoxidized through the electron
transport chain, linked to the formation of ATP..

32. Overview of the Citric Acid Cycle
Energetics
 Oxidation of 3 NADH by ETC coupled with
 oxidative phosphorylation results in the synthesis of
 7.5 ATP. (3 x 2.5 = 7.5)
 FADH2 leads to the formation of 2ATP. ( 1 x 1.5 = 1.5)
 One substrate level phosphorylation GTP.
 Thus, a total of 10 ATP are produced from one acetyl
CoA.

33. Regulation of TCA Cycle
Three regulatory enzymes
1. Citrate synthase
2. Isocitrate dehydrogenase
3. α-ketoglutarate dehydrogenase

34. Significance of TCA Cycle
The citric acid cycle is not only a pathway for oxidation of two-
carbon units, but is also a major pathway for interconversion of
metabolites arising from transamination and deamination of
amino acids, and providing the substrates for amino acid synthesis
by transamination, as well as for gluconeogenesis and fatty acid
synthesis.
• Because it functions in both oxidative and synthetic processes, it
is amphibolic.

35. A) Catabolic role OF TCA Cycle
The citric acid cycle is the final common pathway for the
oxidation of carbohydrate, lipid, and protein because glucose,
fatty acids, and most amino acids are metabolized to
acetyl-CoA or intermediates of the cycle. •
The function of the citric acid cycle is the harvesting of high-
energy electrons from carbon fuels.
• 1 acetate unit generates approximately 12 molecules of ATP
per turn of the cycle.

36. B) Anabolic role of TCA cycle
As a major metabolic hub of the cell, the citric acid cycle
also provides intermediates for biosynthesis of various
compounds.
i) Role in Gluconeogenesis- All the intermediates of the cycle
are potentially glucogenic, since they can give rise to oxaloacetate,
and hence net production of glucose (in the liver and kidney, the
organs that carry out gluconeogenesis).

37. The key enzyme that catalyzes net transfer out of the cycle into
gluconeogenesis is phospho-enol-pyruvate carboxy kinase,
which catalyzes the decarboxylation of oxaloacetate to
phosphoenolpyruvate, with GTP acting as the phosphate donor.

38. ii) Role in synthesis of amino acids
 Aspartic acid is a precursor of Asparagine, Lysine, Methionine,
Threonine and Isoleucine. These amino acid except Asparagine are
essential amino acids, they are synthesized only in plants.

39. iii) Role of TCA cycle in fatty acid synthesis
Role in fatty acid synthesis- Acetyl-CoA, formed from
pyruvate by the action of pyruvate dehydrogenase, is
the major substrate for long-chain fatty acid synthesis .

40. iv) Role in Heme synthesis
Succinyl co A
condenses with amino
acid Glycine to form
Alpha amino beta keto
Adipic acid, which is
the first step of haem
biosynthesis.

41. v) Role in purine and pyrimidine synthesis
 Glutamate and Aspartate derived from TCA cycle are utilized
for the synthesis of purines and pyrimidines.

42. Thank you
for
your
attention!