2. Citric Acid Cycle
– Also known as:
• Tricarboxylic acid (TCA ) cycle
–After its substrates, Citrate &
Iso-citrate
• Krebs cycle
–(After its discoverer, Hans Krebs)
– Special metabolic pathway which runs in
mitochondria
• Most of our cells contain mitochondria which act
as “mobile power plant”
• Few cells however, don’t have mitochondria
–E.g., Red Blood cells (do not run Krebs’ cycle )
• All the enzymes which mediate the cycle
present within mitochondria 2
3. Cont…
Mitochondria:
– Intracellular organelles which are bounded by
double membranes
• Outer membrane
• Inner membrane
– What was origin of mitochondria?
• Originally, mitochondria were energy producing
bacteria
• Millions’ of years back, visited eukaryotic cell
– Eukaryotic cell provided protection & nutrition to
mitochondria
– Bacteria were very happy & provided energy
– What is proof that mitochondria were bacteria?
• Like bacterial DNA, mitochondrial DNA is circular
3
4. Cont…
– Inter-membraneous space
– Matrix
• All the substances in the inner side of inner membrane
– What are substances present in the mitochondrial
matrix?
» Mitochondrial DNA
» Mitochondrial RNA
» Mitochondrial Ribosomes
» Krebs cycle enzyme
» Pyruvate dehaydrogenase complex
» FA beta oxidation enzymes
4
5. Cont…
– What are substances present in the mitochondrial
inner membrane?
» ETCS protein complex
» Mobile electron carriers: Co-Q & cytochrome
C
» Inner membrane particle responsible for ATP
synthesis
» Carrier proteins
5
6. Cont…
– Cyclic process
• Final product of pathway is exactly same to initial
substrate
What is the purpose of Krebs Cycle?
– Concerned with:
• Breakdown of acetyl moiety of Acetyl-CoA
• Provision of intermediates to biosynthetic pathways
• Catabolism of acetyl-CoA:
– Acetyl-CoA is a major fuel for energy producing
cycle (Krebs cycle)
– Passes through cycle & catabolized, while releasing
energy
– From where Acetyl-CoA comes?
– Breakdown product of carbon skeletons of major fuels
in our body (Carbohydrates, Lipids & Proteins) 6
8. Cont…
• What happens when acetyl-CoA moves
through the cycle?
– Two carbon-units (acetyl moieties) of acetyl-CoA
are lost as 2 molecules CO2
– Energy released is captured in the form of 3
molecules of NADH + H+ & one molecule of
FADH2
– NADH + H+ & FADH2 molecules enter into
electron transport chain system (ETCS) to
regenerate NAD+ & FAD
• Purpose of NADH + H+ & FADH2 molecules is to take
energy rich electrons and protons from Krebs cycle to
ETCS
8
9. Cont…
• What will happen when energy rich electrons &
protons are passing through ETCS?
– Oxygen will be added to pathway at end, meets
proton & H2O is produced
– Energy released will be utilized to convert ADP + Pi
into ATP
• In summary, Krebs cycle:
– Final common pathway of catabolism of major body
feuls
– Involves a sequence of dehydrogenation & de-
carboxylation reactions
• De-carboxylation helps blow out CO2
• Dehydrogenation helps transfer protons & electrons
to NAD+ & FAD
– Responsible to produce H2O & to oxidative
phosphorylation of ADP into ATP
9
10. Reactions of Citric Acid Cycle
– Junction Reaction:
• Where is the cellular location of Krebs’ cycle?
=> Mitochondrion
• Where is the cellular location of glcolysis?
=> Cytosol
• How pyruvate can be transported from the cytosol
into the mitochondrial matrix?
=> Pyruvate TOM & TIM
• How pyruvate can be converted into Acetyl-CoA?
=> Oxidative decarboxylation
• Junction reaction between
Glcolysis and Krebs’ cycle
10
14. Clinical Comments related to junction reaction
• Congenital Lactic Acidosis:
– X- linked dominant defect in PDH complex
enzyme system
– Can be of three types:
• Severe
– Neonatal (intra-uterine) death
• Moderate
– Severe mental retardation
– Death in infancy
• Milder
– Persons may survive a little more
– CNS dysfunction during carbohydrate meal
» Failure in coordination of body movement =>
Episodic Ataxia
– Mgt: Ketogenic diet 14
15. Cont…
• Arsenic Poison:
– Arsenate binds with Lipoic acid component of E2
– Produce very mild inhibition
• Progressive accumulation of lactic acidDamage to
CNS
– Mgt: Dimercaprol which binds preferentially to
arsenate than lipoic acid & excreted
15
18. Step-1: Formation of Citric Acid (Condensation
Reaction)
– Oxaloacetate (4-C) condenses with acetyl CoA
(2-C)
– Citrate (6-C compound) is formed
– Catalyzed by citrate synthase
– Thioester bond in acetyl CoA is hydrolyzed
– Hydrolysis of thioster bond drives the reaction
forward
– Irreversible reaction step
18
19. Cont…
Step-2: Formation of Isocitrate (Dehydration
/ Rehydration Reaction)
– Reversible reaction step
– Citrate is isomerized to isocitrate
– Catalyzed by aconitase (Inhibited by Fluoroacetate)
– Two-step process:
• Dehydration of citrate forming cis-aconitate
• Rehydration of aconitate to form isocitrate
19
20. Cont…
Step-3: Formation of Alpha Keto Glutarate
(Oxidative Decarboxylation Reaction)
– Irreversible reaction step
– Isocitrate is oxidatively decarboxylated to α-
keto glutarate
– Catalyzed by isocitrate dehydrogenase
– Two-step process:
• Dehydrogenation of isocitrate forming oxalosuccinate
• Decarboxylation of oxalosuccinate forming alpha keto
glutarate
– One molecule NADH + H+ is generated
– One molecule of CO2 is liberated
20
22. Cont…
Step-4: Formation of Succinyl CoA (Oxidative
Decarboxylation Reaction)
– Irreversible reaction Step
– Alpha keto glutarate is oxi-datively
decarboxylated to succinyl CoA
– Catalyzed by alpha keto glutarate
dehydrogenase Complex
• One molecule NADH + H+ is generated
• One molecule of CO2 is liberated
– Similar reaction mechanism to PDH complex
22
25. Cont…
Step-5: Generation of Succinate (Substrate
Level Phosphorylation Reaction)
– Irreversible reaction step
– Thioester bond of succinyl CoA is broken to form
Succinate & high energy phosphate (GTP)
– Catalyzed by succinate thiokinase (succinyl CoA
synthetase)
• One molecule of GDP is phosphorylated to GTP
• GTP can be converted to ATP by reacting with an ADP
molecule:
– GTP + ADP → GDP + ATP (catalyzed by Nucleoside
diphosphokinase)
– What is difference b/n synthase &
synthetase? 25
27. Cont…
Step-6: Formation of Fumarate
(Dehydrogenation Reaction)
– Reversible reaction step
– Succinate is dehydrogenated to fumarate
(unsaturated dicarboxylic acid)
– Catalyzed by a flavoprotein enzyme, succinate
dehydrogenase
• One molecule of FADH2 is generated
– Succinate dehydrogenase
• Located in the inner side of inner mitochondrial
membrane
• Competitively inhibited by malonate
27
29. Cont…
Step-7: Formation of Malate (Hydration
reaction)
– Reversible reaction step
– Hydration of fumarate to L-malate
– Catalyzed by Fumarase
• Specific to trans-double bond of fumarate & L-isomer of
malate
29
30. Cont…
Step-8: Regeneration of Oxaloacetate
(Dehydrogenation Reaction)
– Reversible reaction step
– Malate is oxidized to oxaloacetate (may be
viewed as a catalyst)
– Catalyzed by malate dehydrogenase
• One molecule of NADH + H+ is generated
30
32. Overall energy balance of Krebs Cycle
• By the help of Krebs cycle:
– Large amount of energy is generated in the form
of ATP via Oxidative phosphorylation
• Accounts for 2/3rd of ATP generated from fuel
oxidation
– Small amount of energy is generated directly
when GTP is converted into ATP via Substrate
Level phosphorylation
• ATP & GTP are Cash energy
• NADH & FADH2 are Check Energy
– Worth of one NADH is 3 ATP & FADH2 is 2 ATP
32
33. Cont…
• Over all reaction equation for one round Krebs
cycle
– Acetyl-CoA+ 2H2O + 3NAD+ + FAD + GDP + Pi
2CO2 + GTP + 3NADH + 3H+ + FADH2 + CoASH
– Fate of the acetyl moiety of Acetyl-CoA
• One molecule of Acetyl-CoA Two molecules of CO2
– Fate of phosphoryl groups
• One molecules of GDP & Pi one molecules of ATP
– Fate of Electrons
• Four pairs of electrons leave cycle in form of 3NADH
& FADH2
• 12 ATP molecules are formed in one round of Krebs
cycle after re-oxidation of 3NADH & FADH2 via ETCS
33
34. Cont…
– Q1: When one molecule of glucose is gone into a
cell, how much ATP will be produced in the
presence of Oxygen?
• Answer:36/38 ATP (How?)
– Hint:
• Glycolysis pathway = 6/8 ATP (why?)
• Junction pathway= 6 ATP
• Krebs’ cycle = 2 ATP via substrate level
phosphorylation & 22 ATP via oxidative
phosphorylation => 24 ATP
34
37. Regulation of Citric Acid Cycle
(1)Respiratory control via ETCS & oxidative
phosphorylation
– Krebs Cycle runs only in presence of Oxygen =>
aerobic pathway
– Respiratory control exerts main control on
activities of Krebs cycle
• E.g., Absence of O2 (anoxia) or partial deficiency of
O2 (hypoxia) causes total or partial inhibition of cycle
37
38. Cont…
(2) Regulation according to energy
requirement of cell
– E.g., During muscular contraction
• Rise in intracellular Ca2+ level:
– Stimulate multiple enzymes of cycle:
» PDH complex (Junction reaction)
» Citrate synthase
» Isocitrate dehydrogenase
» α-Ketoglutarate dehydrogenase
• More ADP,AMP/ATP ratio
– Stimulate multiple enzymes of cycle
• More NAD+/NADH & FAD/FADH2 ratios
– Stimulate multiple enzymes of cycle 38
40. Link b/n TCA cycle & other metabolic pathways
– TCA cycle termed as amphibolic pathway in
nature
• Has dual role
– Catabolic
» Concerned with degradation of Acetyl-CoA to
generate ATP
– Anabolic
» Concerned with provision of intermediates for
biosynthetic pathways
• Biosynthesis of glucose
• Fatty acid synthesis
• Inter-conversion of Amino acids
• Cholesterol & steroid synthesis
40
45. Cont…
– Role of Vitamins in TCA Cycle
• Five vitamin B-complexes are associated with TCA cycle:
– Riboflavin used in:
» FAD form
• Coenzyme for Succinate DH
– Niacin used in:
» NAD+ form
• Coenzyme for ICDH, α-KGDH & MDH
– Thiamine used in:
» TPP form
• Coenzyme for α-KGDH
– Lipoic acid used in:
» Oxidized lipoamide (LS-SL) form & dihydrolipoamide (2-LSH)
form
• Coenzyme for α-KGDH
– Pantothenic acid: Used as part of coenzyme A
» Coenzyme for α-KGDH & Succinate thiokinase 45
