Citric Acid Cycle (TCA cycle, Krebs Cycle)

• Production of Acetyl CoA

• Reactions of Citric Acid Cycle
• Regulation of CAC

Products of the TCA

• ATP (?GTP)
• NADH
• FADH2
• H2O and CO2
 Where do these processes occur?
Geography
• Glycolysis in the cytosol
• Krebs in mitochondrial matrix
• Mitochondrion
– Outer membrane very permeable
• Space between membranes called intermembrane space
– Inner membrane (cristae)
• Permeable to pyruvate,
• Impermeable to fatty acids, NAD
– Matrix is inside inner membrane
Location of cellular respiration
 CAC
• A series of chemical rxns in the cell that
break down food molecules into CO2 and
H2O and energy is produced.
• In plants and animals this takes place in
the mitochondria and in cytosol in bacteria
• Was described by Adolf Krebs 1937,
hence “Kreb’s cycle”
• In order for food to produce most energy it
has to go through the CAC; to enter the
CAC food must be broken down into acetyl
groups i.e the product of stage 1 of
metabolism pyruvate is converted to
acetylcoA

• Acetyl-CoA to water and CO2 is called

cellular respiration
 Cellular respiration
• Cellular respiration is a process by which cells
consume O2 and produce CO2
• It occurs in 3 stages
I. Organic fuels converted to 2C fragments (AC-CoA)
II. Acetyl groups are fed into CAC where it is oxidized
to rls CO2 and the energy released is stored in the
reduced coenzymes; electron carriers(NAD/FAD)
III. Oxidation of the reduced co-enzymes giving up
protons and electrons which are transferred to O2
the terminal e- acceptor. The energy from the e-
transfer is conserved in the form of ATP(Oxidative
phosphorylation)
 Conversion of pyruvate to Acetyl
CoA
NAD+ NADH HSCoA CO2 O
O
O
H3C H3C SCoA
O
pyruvate dehydrogenase complex acetyl CoA

pyruvate

• 2 per glucose (all of Kreb’s)

• Oxidative decarboxylation
• Makes NADH
• -33.4kJ
 PDH complex reactions

Note the 3 enzymes, the coenzymes, the transfer of 2C

units, the production of NADH. What vitamins are involved?
 Fates of Acetyl CoA
O
Kreb's
TAG's H3C SCoA
acetyl CoA CO2, ATP, NADH...energy

no CHO present

ketone bodies

• In the presence of CHO and using energy

– Metabolized to CO2, NADH, FADH2,GTP and, ultimately, ATP
• If energy not being used (Lots of ATP present)
– Made into fat
• If energy being used, but no CHO present
– Starvation
– Forms ketone bodies (see fat metabolism slides)
– Danger!
 Kreb’s Cycle
acetyl CoA
H3C SCoA
C
O O
O
CoASH C
O O O H2O
O + CH2 O
C NADH C
NAD
C O
HO C C O
HC OH citrate synthase CH2
CH2
H 2O CH2 malate C citrate
dehydrogenase
C O O
O O C O
O
fumarase O
C O oxaloacetate
C H malate
aconitase
H C fumarate

O
C
O Kreb's Cycle O O
FADH2 C
succinate
dehydrogenase HO CH O
HC C O
FAD alpha ketoglutarate CH2
O SCoA O C isocitrate
O GTP
NADH O O
C CoASH C
NAD C NADH O O
CH2 CH2 C O CO2 NAD
CH2 GDP CH2
CH2 CoASH
C C CO2 CH2
succinyl CoA isocitrate dehydrogenase
O O synthetase O O alpha ketoglutarate C
succinate succinyl CoA
dehydrogenase
O
O
 Net From Kreb’s
• Oxidative process
– 3 NADH
– FADH2
– GTP
• X 2 per glucose
– 6 NADH
– 2 FADH2
– 2 GTP
• All ultimately turned into ATP (oxidative
phosphorylation…later)
 Citrate Synthase Reaction (First)
O O O
C O
C
C O H2 O CoASH
CH2 O
H3C SCoA CH2
C C HO C C O
O + O
O
citrate synthase CH2
C
O O
acetyl CoA oxaloacetate
citrate

• condensation
• -32.2kJ
 Aconitase Reaction
O O O O
C C
CH2 O HO CH O
HO C C O HC C O
CH2 CH2
aconitase
C C
O O
O O

citrate isocitrate

• Forms isocitrate
• Goes through alkene intermediate (cis-aconitate)
– elimination then addition
• Hydroxyl moved and changed from tertiary to secondary
– (can be oxidized)
• 13.3kJ
 Isocitrate Dehydrogenase
O O
O O C
C C O
HO CH O
NAD NADH CO2 CH2
HC C O CH2
CH2
C C
O O isocitrate dehydrogenase O O

isocitrate alpha ketoglutarate

• All dehydrogenase reactions make NADH or FADH2

• Oxidative decarboxylation
• -20.9kJ
• Energy from increased entropy in gas formation
 α-ketoglutarate dehydrogenase
O O
C SCoA O
C O
C
CH2 CO2 NADH
CoASH NAD CH2
CH2
CH2
C C
O O O O
alpha ketoglutarate
dehydrogenase
alpha ketoglutarate succinyl CoA

• Same as pyruvate dehydrogenase reaction

• Formation of thioester
– endergonic
– driven by loss of CO2
• increases entropy
• exergonic
• -33.5kJ
 Succinyl CoA synthetase
SCoA O O
O
C C
CH2 GTP CoASH CH2
GDP
CH2 CH2
C C
O O succinyl CoA O O
succinyl CoA synthetase
succinate

• Hydrolysis of thioester
– Releases CoASH
– Exergonic
• Coupled to synthesis of GTP
– Endergonic
– GTP very similar to ATP and interconverted later
• -2.9kJ
 Succinate dehydrogenase
O O O
O
C FADH2 C
CH2 FAD C H
CH2 H C
C succinyl CoA
C
O O
O O dehydrogenase

succinate fumarate

• Dehydrogenation
• Uses FAD
– NAD used to oxidize oxygen-containing groups
• Aldehydes
• alcohols
– FAD used to oxidize C-C bonds
– 0kJ
 Fumarase
O O O O
C H2 O C
C H HC OH
H C CH2
C fumarase
C
O O O
O
fumarate
malate

• Addition of water to a double bond

• -3.8kJ
 Malate Dehydrogenase
O O
C O O
HC OH C
NAD NADH C O
CH2 CH2
C C
O O malate O
dehydrogenase O
malate
oxaloacetate

• Oxidation of secondary alcohol to ketone

• Makes NADH
• Regenerates oxaloacetate for another round
• 29.7 kJ
 Net From Kreb’s
• Oxidative process
– 3 NADH
– FADH2
– GTP
• X 2 per glucose
– 6 NADH
– 2 FADH2
– 2 GTP
• All ultimately turned into ATP (oxidative
phosphorylation…later)
 Total Energy per glucose
• Cytosol
– Glycolysis
• 2 NADH
• 2 ATP
• Mitochondrion
– Pyruvate dehydrogenase
• 2 NADH
• Krebs
– 6 NADH
– 2 FADH2
– 2 GTP
 Total Energy/glucose
• In mitochondrion:
– Each NADH makes 2.5 ATP
– Each FADH2 makes 1.5 ATP
– GTP makes ATP
• So…
– From in mitochondrion
• 8 NADH X 3 (2.5) ATP/NADH = 24 ATP
• 2 FADH2 X 2(1.5) ATP/FADH2= 4 ATP
• 2 GTP X 1 ATP / GTP = 2 ATP
• TOTAL in mitochondrion 30 ATP
 Total Energy/ glucose
• Cytosol
– 2 ATP
– 2 NADH
• NADH can’t get into mitochondrion
• In eukaryotes two pathways,
– transferred to FADH2
» get 2 ATP/ FADH2
– Or transferred to NADH
» Get 3 ATP/ NADH
– (Not a problem in prokaryotes (why?))
– 2 NADH X 2 (1.5) ATP = 4 ATP
– Or 2 NADH X 3 (2.5) ATP = 6 ATP
» + =2 ATP
» Total 3+ 2 or 5 + 2 so either 5 or 7
 ATP/glucose
• Ideal / theoretical output in Eukaryotes
– Mitochondrial: 30 ATP
– Cytosolic: 6 or 8 ATP
– Total 36 or 38 ATP/glucose

– 30 ATP X 7.3kcal X 4.18 kJ = 915 kJ

ATP kcal

If 32 ATP = 976 kJ
• Prokaryotes
– 32 ATP X 7.3kcal X 4.18 kJ = 976 kJ
ATP kcal
Other functions of the TCA
 ETC

• Note the location of the various process

 ETC enzyme complexes

The relationship of the enzymes on the cell membrane. Note the tranfer of electrons,
protons and the ATP synthase
Composition of the ETC
enzyme complexes
ETC enzyme complex inhibitors
• Most important known inhibitors:
– Amytal – complex I - a barbiturate
– Rotenone – complex I - plant product
(insect/pesticide)
– Antimycin A – complex III - Antibiotic, from
streptomyces
– Carbon Monoxide CO complex IV and Hb–
50% of poisoning deaths world wide
– Cyanides – Complex IV - smoke inhalation
from industrial, residential fires
– Azides – Complex IV - used in airbags,
preservation of serum, explosive industry
 ATP synthase complex V
• Provides energy for the cell to use through
the synthesis of ATP:
• ATP synthase + ADP + Pi → ATP Synthase
+ ATP
• Energy used in the form of
protium(H+)/protons moving down the
electrochemical gradient from the inter-
membrane space into the matrix of the
mitochondria

• Fo aka oligomycin binding

• Is located within the mitochondria
• Fo (fraction oh)portion is within
the membrane
• F1 (fraction 1)portion of the ATP
synthase is above the membrane,
inside the matrix of the
mitochondria
fraction is inhibited by oligomycin
• These fractions consist of
different protein subunits
• Fo is a proton pore
• F1 is associated with the
synthesis of ATP
ATP synthase
• ATP
synthase
activity is
reversible
 Work to Do
• Regulation of CAC
• CAT this Friday (9th March, 2012)

A man is wise with the wisdom of his time

only, and ignorant with its ignorance
(HENRY DAVID THOREAU)
• Thank You
A man is wise with the wisdom of his time
only, and ignorant with its ignorance
(HENRY DAVID THOREAU)
• Thank You
Citric Acid Cycle (TCA)
3rd Year Biochemistry
School of Medicine
Juba University

Dr Akello