2. • Tricarboxylic Acid (TCA)
cycle (citric acid cycle)
– Also called Krebs cycle
after Sir Hans Kreb.
– is a common pathway for
oxidation of all fuel
molecules.
– Oxidizes two-carbon units
from acetyl CoA (the
common end product of
fuel molecules) in to two
CO2 by capturing all the
energy available from
acetyl CoA as 1 GTP, 1
FADH2 and 3 NADH.
2
3. • TCA cycle cont’d…
– It has eight reaction
that takes place in
mitochondrial matrix
and
– All its enzymes exist in
mitochondrial matrix
except succinate
dehydrogenase.
• w/c exist attached to
inner mitochondrial
membrane.
• Succinate
dehydrogenase is a
common enzyme for
TCA cycle and ETC
acts as a link
between this two
pathways.
3
4. • TCA cycle cont’d…
– TCA cycle is so named
in this pathway
oxaloacetate is
in rxn 1 and regenerated
in rxn 8 and recycled.
– Since oxaloacetate is
recycled; required in
amount to metabolize
large amount of acetyl
CoA, hence acts as an
activator.
– Reactions 1,3,4 are
irreversible and are
regulatory.
4
5. • TCA cycle cont’d…
– TCA cycle produces
of the reduced
coenzymes (FADH2 &
NADH.H+) from
oxidation of glucose.
– w/c are finally used for
the generation of ATP in
the ETC in w/c;
• 1 NADH gives 3 ATPs
• 1 FADH2 gives 2
• And regenerated to
their oxidized forms
(NAD+ and FAD)
respectively
– Using O2 as final
acceptor w/c itself
reduced to H2O
• Generating much of
the ATP available
from oxidation of
molecules.
5
6. • TCA cycle cont’d…
– Though TCA cycle is
oxidative pathway.
• It does not use
oxygen directly in
any of its reactions.
• But requires
ETC/oxidative
phosphorylation to
operate (that
require O2 as the
final electron
acceptor) for re-
oxidation of the
reduced
coenzymes.
• W/c serve as co-
enzymes of the
oxidative steps in
TCA cycle.
6
7. • TCA cycle has both energy
production (catabolic) and
and biosynthetic (anabolic)
function i.e.,
– In addition to
generation of energy, it
provides precursor
molecules for the
synthetic pathways.
– Therefore it is an
amphibolic pathway.
7
8. • Reaction steps of the TCA cycle:
– 1. Synthesis of citrate (6C) from acetyl CoA (2C) and
oxaloacetate (4C) by citrate synthase.
• It is an aldol condensation reaction that form a C-C
bond between the methy carbon of the 2C acetyl CoA
and carbonyl carbon of the 4C dicarboxylic acid
oxaloacetate forming the 6C tricarboxylic acid citrate.
citrate.
– The breaking down of the thioester bond of
acetyl CoA provides the energy for the
endergonic C-C condensation.
• It is an irreversible regulatory step:
8
9. • Reaction steps of the TCA cycle cont’d…
– 2. Isomerization of citrate to isocitrate by aconitase
• First citrate is converted to cis-aconitate
by a dehydration step
• which then converted to isocitrate by a hydration
step with a net result of exchange of H and OH
groups.
– This isomerization converts the tertiary alcohol
alcohol group of citrate, at its 3rd carbon, w/c
is resistant to oxidation to a secondary alcohol
in isocitrate w/c is easily oxidizable.
– Moreover the isomerization helps to form the
chiral intermediate isocitrate
» w/c make the 2 carbons of acetyle CoA
preserved and release the 2 COO- of
oxaloacetate as 2 CO2.
9
10. • Reaction steps of the TCA cycle cont’d…
– 3. Oxidative decarboxylation of isocitrate to α-
ketoglutarate by isocitrate dehydrogenase.
• Involves oxidation of alcohol (OH) group of
isocitrate to keto (R=O) group of α-ketoglutarate.
• And decarboxylation of the COO- at C-3 (β-
carbon).
– Such enzymes catalyzing oxidative decarboxylation
of COO- at β-carbon require metal ions as a
cofactor
– And isocitrate dehydrogenase require Mg2+
or Mn2+.
• It yields the first of three NADH molecules
by the cycle, and releases the first CO2.
• It is also an irreversible and regulatory step:
10
11. • Reaction steps of the TCA cycle cont’d…
– 4. Oxidative decarboxylation of α-ketoglutarate to
succinyl CoA by α-ketoglutarate dehydrogenase
complex.
• It produces second NADH and releases the
CO2.
• It is one of the three α-ketoacid dehydrogenase
complex enzymes. The other 2 are:
– pyruvate dehydrogenase complex and
– branched chain α-ketoacid dehydrogenase
complex.
• All the three require the same five coenzymes.
• It is the third irreversible regulatory step:
11
12. • Reaction steps of the TCA cycle cont’d…
– 5. Cleavage of succinyl CoA to succinate andCoASH
succinate thiokinase (succinyl CoA synthetase).
• It cleaves the high energy thioester bond of
CoA.
• This reaction is coupled to phosphorylation of
GTP that traps the energy released by cleavage of
high energy thioester bond.
• Hence it is the only substrate level
reaction of the cycle.
– Note: GTP & ATP are interconvertable by
nucleoside diphosphate kinase and are
energetically equivalent:
12
13. • Reaction steps of the TCA cycle cont’d…
– 6. Oxidation of succinate to fumarate by succinate
dehydrogenase.
• Succinate dehydrogenase is a flavoprotein containing
FAD as a prosthetic group
• It produces the reduced coenzyme FADH2.
– FAD rather than NAD+, is the electron acceptor
the reducing power of succinate is not sufficient to
reduce NAD+.
– 7. Hydration of fumarate to malate by fumarase or
hydratase.
• Fumarase catalyzes addition of water to the trans
double bond of fumarate to produce L-malate.
• This helps to rearrange the carbons to oxaloacetate.
– 8. Malate is oxidized to regenerate oxaloacetate by
malate dehydrogenase.
• This produces the third and final NADH of the cycle.
• The reaction is endergonic but the removal of
oxaloacetate by the irreversible citrate synthase
reaction derives the reaction forward.
13
15. Energy Yield From TCA cycle
Reaction Yield per per
Acetyl coA
No. of ATP
Isocitratedehydro
genase
1NADH 3
α-Keto glutarate
dehydrogenase
1NADH 3
Succinate
thiokinase
1GTP 1
Succinate
dehydrogense
1FADH 2
Malate
dehydrogense
1NADH 3
Total 12
15
16. • Regulation of TCA cycle
– The primary control sites are the allosteric enzymes that
catalyze the irreversible steps of the cycle.
– These are:
• Citrate synthese (Rxn 1)
• Isocitrate dehydrogenase (Rxn 3) and
• α-ketoglutarate dehydrogenase (Rxn 4).
16
17. • Regulation of TCA cycle cont’d…
– 1. Citrate synhthase.
• Activated by Ca2+ and ADP
,
• Inhibited by ATP
, NADH, SuccinylCoA, and fatty
acylCoAs.
• However the enzyme is primarily regulated by the
availability of its substrates in the liver.
– 2. Isocitrate dehydrogenase
• Activated by ADP (a low-energy signal) and Ca2+,
• Inhibited by ATP and NADH, w/c both are indicators of
availability of abundant energy stores in the cell.
– 3. α-ketoglutarate dehydrogenase complex.
• Activated by ADP
, Ca2+
• Inhibited by ATP
, GTP
, NADH, and succinylCoA.
• However, unlike PDH complex it is not regulated by
phosphorylation/dephosphorylation.
• Note that: in general the rate of the cycle is reduced
when the cell has a high level of ATP but activated by
low level of ATP or high level of ADP
.
17
19. • Electron Transport Chain:
– Is the final stage of oxidation of fuel molecules. It
completes oxidation of glucose.
– It leads to the production of ATP by oxidizing the
reduced potentials NADH & FADH2 (generated by
glycolysis, PDH complex and TCA cycle) to their oxidized
oxidized forms (NAD+ & FAD) respectively.
• Uses molecular oxygen as the final electron acceptor
acceptor w/c is reduced finally to H2O.
• Energy released during transfer of electrons from one
one acceptor to the other used to phosphorylte ADP
ADP to ATP.
– This is referred to as oxidative phosphorylation.
– Note that:
• 34 of the 38 ATPs generated by complete oxidation
of 1 mol of glucose are produced by oxidative
phosphorylation.
• Only 4 are formed by substrate level phosphorylation.
19
20. • Components of the ETC:
– Electron transport chain is composed of four protein
complexes with oxidoreductase enzyme action
arranged in sequence/chain in the inner mitochondrial
membrane as follows:
• Complex I (NADH-Q oxidoreductase)
• Complex II (Succinate-Q reductase)
– It is succinate dehydrogenase enzyme of TCA cycle
– It is the only TCA cycle enzyme exist attached to
inner mitochondrial membrane.
– It is the only complex that does not act as proton
pump
– It serves simply as a physical link b/n TCA cycle and
ETC
• 3) Complex III (Q-cytochrome C oxidoreductase or
cytochrome reductase)
• 4) Complex IV (Cytochrome C oxidase)
20
21. • Components cont’d…
– In addition to the four complexes ETC also contains
electron carriers:
• Cytochrom C and Coenzyme Q (ubiquinone).
– Cytochrom C is a small water soluble heme protein
located loosely bound to the outer surface of the
mitochondrial membrane.
– Coenzyme Q (CoQ) is a mobile lipid soluble
(hydrophobic) electron carrier embedded in the
membrane of mitochondria.
21
22. • Flow of Electrons in ETC
– The complexes and
electron carriers serve
as electron transporters
to O2.
– They are arranged in
sequential order from
the least to highest
standard redox
potential (E’o) i.e. from
most reduced to most
oxidized.
– As electrons flow from
the reduced potentials
through ETC, protons
are pumped through
complex I, III and IV.
22
23. • ATP synthase (Complex V):
– Is enzyme that catalyze the “coupling” of flow of electrons
to O2 (ETC) & ATP synthesis (oxidative phosphorylation).
– It is a large protein complex composed of two subunits
designated as F1 and Fo; F stands for coupling factor.
• F1 is a peripheral membrane protein protruded in to the
matrix side of mitochondria.
– Itis the catalytic sub unit which catalyzes the actual
production of ATP from ADP and Pi.
• Fo is a hydrophobic integral membrane protein that spans
the mitochondrial inner membrane.
– It is the proton conducting unit (proton channel)
through w/c protons pumped out during electron flow
return back to the mitochondrial matrix.
» This releases the energy for ATP synthesis.
23
24. • Chemiosmotic Theory:
– Proposed by Peter Mitchell in 1961 and describes how the
flow of electrons through the electron transport chain (an
endothermic process) is coupled with ATP synthesis (an
exothermic process).
– According to this hypothesis, the two are coupled by a proton
gradient (proton motive force=PMF) across inner
mitochondrial membrane i.e,.
• When protons are pumped out of the mitochondrial
matrix to the intermembrane space during the electron
transport,
– the intermembrane space becomes more positively
charged and acidic than the matrix side
– the matrix becomes more negatively charged & basic.
– Therefore an electrochemical (charge and pH) gradient is
generated across mitochondrial inner membrane.
• This charge & pH imbalance is called proton-motive force
(PMFs) that conserves energy released during the electron
transport.
24
25. • Chemiosmotic Theory cont’d…
– The proton-motive force derives protons to move back into the
matrix to balance the charge and pH imbalance.
– Thus, the free energy that was stored as PMF is released when
protons flow passively back into the matrix through a proton
proton pore within Fo of ATP synthase.
– The free energy released is utilized by ATP synthase (complex
(complex V) to synthesize ATP.
• Note: Protons can pass only through Fo because inner
mitochondrial membrane is impermeable to them.
– Mitchell used the term “chemiosmotic” to describe
enzymatic reactions that involve, simultaneously, a chemical
reaction (chemi) and a transport process (osmotic).
25
