Overall view of energyyielding

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Overall view of energy-yielding central pathway
in the eukaryotic cell Common fuel input to
TCA is acetyl-CoA amino acid, fat, sugar
pathways converge on this metabolite
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The Central Role of the Citric Acid Cycle in
Metabolism
Map at http//www.sigmaaldrich.comx/img/assets/42
02/MetabolicPathways_updated_4.19.05.pdf
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In Eukaryotes, Citric Acid Cycle Occurs in the
Mitochondria

• Glycolysis occurs in the cytoplasm
• Pyruvate is translocated into the mitochondria
• PDH and the citric acid cycle occur in the
  mitochondrial matrix
• Oxidative phosphorylation occurs in inner
  mitochondrial membrane

One enzyme of the cycle, succinate
dehydrogenase, is located in the inner membrane
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Overview Acetyl-CoA synthesis reaction by PDH
Oxidative decarboxylation irreversible NADH
gives up hydride ion to electron transport
chain Each NADH ultimately generates 2.5 ATP
in oxidative phosphorylation
Note high-energy thioester bond
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Pyruvate Dehydrogenase Complex (PDH)

• PDC is a large (Mr  7.8  106 Da) multienzyme
  complex
• pyruvate dehydrogenase (E1)
• dihydrolipoyl transacetylase (E2)
• dihydrolipoyl dehydrogenase (E3)
• short distance between catalytic sites allows
  channeling
• of substrates from one catalytic site to
  another
• channeling minimizes side reactions
• activity of the complex is subject to regulation
• Multiple copies of each E1, E2, E3 per particle

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Cryo-EM reconstruction of the PDH isolated from
bovine kidney
E1 binds TPP
E3 binds FAD

• Modular subunit structure of E2
• Lipoate cofactor attaches to E2
• Long, flexible lipoate arm is the
• key to PDH activity

• Overall Diameter 50 nm (500 Å)
• 60 x E2 subunits core (D 25 nm)
• Regulatory enzymes also attach
• (kinase and phosphatase)

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Lipoic acid cofactor Two thiol groups undergo
reversible oxidation to disulfide Also
functions as a carrier of hydrogen or acetyl
groups Covalently attached through lysine of E2
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Pyruvate Dehydrogenase Complex

• 5 cofactors (1) thiamine pyrophosphate (TPP),
  (2) FAD,
• (3) CoA-SH, (4) NAD, (5) lipoic acid
• 3 enzyme activities
• Similar to a-ketoglutarate dehydrogenase complex
  (TCA)

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Pyruvate decarboxylase and PDH in each case R1
-CH3 Hydroxyethyl-TPP is the covalently modified
cofactor in each case
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• PDH complex works by
• substrate channeling on
• the surface of the large
• particle
• Substrate and reducing
• equivalents are carried
• on lipoate

Step 1 Decarboxylation of pyruvate to
hydroxyethyl (TPP, E1 slow step) Step 2
Oxidation of hydroxyethyl to a carboxylic acid
by E1 (lipoic acid S-S from E2 is reduced to
two SH then linked to acetate) Step 3 Swinging
lipoyl arm of E2 delivers the 2C group to the E2
active site, transesterifies it to CoA,
releases free acetyl-CoA, and delivers the
reduced lipoyllysine to E3 Step 4 Reoxidation
of lipoamide cofactor electrons to FAD Step 5
Regeneration of oxidized FAD electrons to NAD,
form NADH
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The Citric Acid Cycle

• 8 enzymes in a cyclic pathway
• CO2 released at steps 3 and 4
• these carbons are not from
• acetyl-CoA
• Succinate and fumarate are
• symmetric can no longer
• denote where the acetate is
• Electrons passed to NAD
• and to FAD one substrate-
• level phosphorylation
• Steps 1, 3, 4 irreversible all
• others reversible
• No net use of oxaloacetate
• concentrations of this
• metabolite are kept low
• TCA intermediates are also
• metabolic precursors, and
• anaplerotic reactions must
• be used to replenish these.

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Reactions of the TCA cycle Steps 1, 3, 4 are
essentially irreversible other steps are
reversible

• Step 1 C-C bond formation forms citrate
• Step 2 Isomerization via dehydration -
  hydration
• Steps 3-4 Oxidative decarboxylations to give 2
  NADH
• Step 5 Substrate-level phosphorylation to give
  GTP or ATP
• Step 6 Dehydrogenation to give reduced FADH2
  (ultimately QH2)
• Step 7 Hydration
• Step 8 Dehydrogenation to give NADH

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Citrate synthase reaction
unliganded
substrates bound (induced fit)
Driven by hydrolysis of high-energy
thioester Only cycle reaction with C-C bond
formation this is a Claisen condensation Essentia
lly irreversible process Oxaloacetate must bind
first (ordered) for induced-fit
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Aconitase step 2
Mechanism proton abstraction from C2 from
citrate (pink) by a catalytic base double
bond formation, elimination of water
facilitated by one iron of the Fe-S
center Reattack by water is also at C2 to
promote isomerization Reattack could yield
citrate or isocitrate, but isocitrate is depleted
in next step Fe promotes water ionization by
lowering its pKa. Rare example of iron-sulfur
cluster involved in a non-redox
reaction Cytosolic isoform also contains Fe-S
cluster. If Fe low, Fe-S disassembles and
activity is lost. Apo-enzyme binds and
stabilizes mRNA for iron-uptake protein.
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How to Distinguish between Two CH2-COO- Arms?
Citrate is a prochiral molecule (no chiral
center, but able to react asymmetrically in an
asymmetric enzyme active site) Three attachment
points are needed for stereospecific abstraction
of a hydrogen
a1
d
c
a2
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Oxidative decarboxylation steps (3 4) in the
TCA cycle
transient intermediate stabilized by manganese
DG -21 kJ/mol
Mechanism of a-KG dehydrogenase is very
similar to the PDH complex
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(No Transcript)
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Substrate-Level Phosphorylation-Succinyl-CoA
synthetase

• Different isozymes use ADP vs. GDP
• High energy thioester converted to
• ATP conserves high-energy bond
• DG -2.9 kJ/mol
• Phosphorylated histidine also conserves
• bond energy in an E-S covalent complex
• intermediate
• GTP ADP ? GDP ATP (DG 0 NDP kinase)

Pi
Release of CoA
Transfer to GDP
Formation of phospho-His
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Step 6 Oxidation of succinate to fumarate
Enzyme bound tightly on inner mitochondrial
membrane Electrons from succinate go to FAD,
then to Fe-S clusters Electrons are then
funneled to Coenzyme Q and eventually to
oxygen in oxidative phosphorylation
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Step 7 Fumarate hydration to malate Reversible
highly specific for trans isomer in forward
rxn. highly specific for L-malate in reverse
rxn.
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Last (8th step of TCA cycle) Highly unfavorable
in standard-state driven by highly favorable
citrate synthase rxn. (step 1) concentration
of oxaloacetate is very low in cells
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Energy yield is in the form of ATP, NADH and FADH2
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Energy yields each NADH yields 2.5 ATP in ox.
phos. each FADH2 yields 1.5 ATP in ox.
phos. glycolysis PDC TCA cycle ? 30-32 ATP
per glucose 32 ATP (30.5 kJ/mol) 976 kJ/mol
(34 of 2840 kJ/mol) Under cellular conditions
efficiency is 65