TCA Cycle

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TCA Cycle
most active of all metabolic pathways
L. William Stillway, Ph.D. Medical University of
South Carolina Charleston, SC 29425
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The TCA Cycle is Amphibolic

• Catabolic (oxidation)
• Decarboxylates metabolites to CO2.
• Generates GTP
• Generates reduced coenzymes.
• NADH
• FADH2
• Provides succinyl CoA for oxidation of ketone
  bodies
• Anabolic (synthesis)
• Fatty acids
• Glucose (gluconeogenesis)
• Amino acids
• Porphyrins (heme)

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Location

• Present in virtually all cells (RBC excepted)
• Mitochondrial Matrix

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Watch for These

• Steps involving NAD and FAD
• Step involving substrate-level phosphorylation
• Steps that liberate carbon dioxide
• Number of carbons at each step
• Regulation
• Alternate sources of carbon (other than acetate)

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TCA Oxidative Summary
Acetyl CoA (2C)
2CO2
3NAD
3NADH
FAD
FADH2
GTP
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TCA Anabolic Summary
Input
Output
Fatty Acids Steroids
Cit
Acetyl CoA
OAA
Asp
Mal
Glc
a-KG
Glu
Succ CoA
Met, Ile, Val
Porphyrins (Heme)
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Ionization Nomenclature of Acids

• For our purposes the names of acids will be kept
  short.
• For example, oxaloacetate will be used to
  represent
• any state of ionization of oxalacetic acid.

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Mitochondrion
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Getting to the TCA, I
Pyruvic Acid
Transporter
Pyruvic Acid
Pyruvic acid must be transported into the
mitochondrion by a transport protein. Note that
it is transported in a neutral form, meaning that
it carries a proton with it.
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TCA cycle reactions
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TCA cycle summary
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Getting to the TCA, II
Pyruvic Acid
CO2 Fixation
Oxidative Decarboxylation
Pyruvate Dehydrogenase
Pyruvate Carboxylase
CoASH
TPP, CoA, Lipoate, FAD, NAD
CO2
Biotin
NADH
CO2
Acetyl CoA
Oxaloacetic Acid
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The Pyruvate Dehydrogenase Complex (PDC)
Also called Pyruvate Dehydrogenase or PDH

• Contains five enzyme proteins

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Channeling, Tunneling, and Metabolons
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PDC is an example of channeling

• Channeling is a metabolic assembly line

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Lipoate and Lipoamide
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E1
E2
CoASH
NAD
FADH2
E3
FAD
NADH
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E1
E2
CoASH
NAD
FADH2
E3
FAD
NADH
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PDC of E. coli
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Branched-chain amino acids and maple syrup urine
disease (MSUD)
Transaminases convert the a-amino groups to an
a-keto group.
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MSUD Contd
MSUD is caused by defective E1 in the a-keto acid
dehydrogenases which results in accumulation of
a-keto acids and BCAA.
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Pyruvate and the TCA Cycle
The TCA cycle begins with acetyl CoA, not
pyruvate Pyruvate is a precursor to acetyl
CoA Pyruvate is a precursor to oxaloacetic acid
(OAA) OAA is required to burn acetyl CoA Energy
demand - more OAA provided OAA also plays role
in Fatty acid biosynthesis Ketone body
metabolism
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PDC is Regulated by Covalent Modification
High Energy Charge -- phosphorylation of PDC
Phosphorylation inhibits PDC
High ratios of ATP/ADP NADH/NAD Acetyl
CoA/CoASH
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Insulin stimulates PDC
Insulin
Citrate
PDC
Pyruvate
Acetyl CoA
Citrate
Fatty Acids
Pyruvate
OAA
Acetyl CoA
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Nutritional State Regulates PDC

• Starvation
• Diabetes type 1
• Low carbohydrate diets
• These all induce the production of the PDC kinase
  protein via mRNA.
• The kinase, which is more active, inhibits E1,
  and PDC activity.
• This slows the rate of fuel oxidation. (Maybe
  this is why exercise is so important.)

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Circle these reactions in the next slide

• Oxidative steps that produce NADH
• Oxidative step that produces FADH2
• Decarboxylation steps
• Substrate-level phosphorylation

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Two decarboxylations
Four oxidations
3 make NADH 1 makes FADH2
One P generated
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TCA reaction details
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1. Citric Acid from AcCoA OAA
Driven by breakage of thioester linkage
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2. Isomerization of Citrate
Aconitate is enzyme-bound. Citrate is
symmetrical. Isocitrate is asymmetrical. Citrate
binds to aconitase asymmetrically.
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3.

• Oxidative Decarboxylation
• Produced
• NADH
• CO2
• a-ketoglutarate

Isocitrate
ADP
NAD
NADH
NAD
ATP
NADH CO2
NADH CO2
Important Control Point M- ATP, NADH M ADP
a-Ketoglutarate (a -KG)
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Isocitrate Dehydrogenase the main regulatory
enzyme in the TCA
Fatty Acids Cholesterol M- PFK1
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4. Oxidation of a-ketoglutarate
Transaminase
Dehydrogenase
???SH
NAD
Oxidative Decarboxylation Analogous enzyme
complex to pyr dehydrog. (3 enzymes) Produced
Succinyl CoA (Hi E) ?O2 NADH
?O2
NADH
????????????
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Transamination mechanism
a-KG
Glu


pyridoxal phosphate
Ala
Pyruvate
In this example, a-KG is transaminated with
alanine to produce glutamate and pyruvate. The
amino group is exchanged for a keto group. The
reaction is freely reversible. Ala was just an
example almost any of the 20 amino acids could
have been used, but they all transaminate with
the a-KG/glutamate combination.
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Succinyl CoA Importance

• Succinyl CoA contains a high-energy thioester
  linkage.
• Succinyl CoA can be made from
• apha-ketoglutarate
• methionine
• isoleucine
• valine
• propionyl CoA
• Conversion of Met, Ile, Val and propionyl CoA to
• succinyl CoA all require the coenzyme form of
  B12.
• Succinyl CoA is the source of carbons for
  synthesis of heme.

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Generation of PConservation of energy from
Hi-Energy Thioester
5.
??Gof succinyl CoA hydrolysis -9.0kcal.mol-1)
GDP Pi
Succinyl CoA Synthase
GTP CoASH

ATP
ADP
GDP
Nuceoside Diphosphokinase
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6. Succinate Dehydrogenase
This enzyme is a flavoprotein. Part of the inner
mitochondrial membrane. (Complex II, succinateQ
reductase) FAD and iron-sulfur complexes (Fe S)
are prosthetic groups. Succinate and fumarate are
symmetrical.
FAD
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7. Hydration of fumarate
Water is added across the double bond. Malate is
asymmetrical.
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8. Regeneration of OAA
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Fates Sources of OAA
Pyruvate
No transport of OAA through the IMM
Proteins
Fatty acids
Cit
Asp Asn
Cit
OAA
(TCA)
Food
Malate
Gluconeogenesis
Malate
Glc
OAA
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Transamination mechanism
a-KG
Glu


pyridoxal phosphate
OAA
Asp
In this example, a-KG is transaminated with
aspartate to produce glutamate and OAA. The amino
group is exchanged for a keto group, and the
enzyme is aspartate transaminase (AST), one of
the enzymes measured to assess liver function.
The reaction is freely reversible. Again, almost
any of the 20 amino acids can be
transaminated, and they all transaminate with the
a-KG/glutamate combination.
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Energy Yield of the TCA
Isocitrate
3
NADH
a-KG
3
NADH
Succinate
2
FADH2
Malate
3
NADH
11P
Substrate-level phosphorylation
1
GTP
12 P (ATP) per turn
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A few points of emphasis
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Substrate-Level Phosphorylationsynthesis of p
with a high-energy intermediate
The free energy in the thioester linkage is
conserved as a pyrophosphate linkage in GTP.
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Pi Supply
Synthesis of GTP requires Pi
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Activity of the TCA cycle is dependent on the
supply of acetyl CoA and oxaloacetic acid.
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Regulation of TCA
High energy charge inhibits
Low energy charge stimulates
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Allosteric Modifiers of TCA Enzymes
Citrate synthase
M- ATP (bacteria)
Isocitrate dehydrogenase
M- ATP M ADP, NAD
a-ketoglutarate dehydrogenase
M- ATP, Succinyl CoA, NADH
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Coupling of the TCA cycle to electron transport,
ATP production, and oxygen uptake
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Measuring O2 uptake
O2 electrode
Introduction tube
Sealed chamber
Mitochondria Substrate Pi ADP
Recording device
Stirring bar
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Respiratory ControlOxygen uptake is tightly
coupled to ADP
156 mm Hg
pO2
Time
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PO ratio
The number of Pi incorporated into ADP per oxygen
atom utilized.
(Not the number of Pi incorparated per O2)
i.e., the number of molecules of ATP produced per
oxygen atom
Example What is the PO ratio if 1.5µM of O2 is
consumed in the conversion of 3.0µM of ADP to
ATP? Ans 1
3.0µM ADP
1
(1.5µM O2 x 2)
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Anaplerotic Reactions
Anaplerotic
Since there is no net synthesis of 4-carbon
intermediates the cycle will need carbons added
to it if an intermediate is used for synthesis.
Examples
Citrate from diet OAA from Asp or from
pyruvate Succinyl CoA from propionyl
CoA a-ketoglutarate from glutamate
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Pyr
AcCoA
AcCoA
CO2
Glc
Glc
Cit
OAA
OAA
Cit
OAA
Mal
Mal
Mal
Isocit
Isocit
CO2
CO2
Fum
Fum
a-KG
a-KG
CO2
CO2
SuccCoA
Succ
SuccCoA
Succ
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Glc
Lactate
Ala
More Anaplerotic Reactions
Pyr
Pyr
Pyr
Pyr
AcCoA
Glc
Cit(food source)
OAA
OAA
OAA
Glc
Cit
Cit
Glc
Isocit
Isocit
Mal
Mal
Mal
Glu
a-KG
a-KG
a-KG
Met,Val,Ile
Met,Val,Ile
Fum
ALA
ALA
Heme
SuccCoA
SuccCoA
SuccCoA
SuccCoA
SuccCoA
Succ
ALA
ALA
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CH2COOH
Citric Acid
A symmetrical compound No asymmetric center
OH
CH2COOH
HOOC

• Tetrahedron
• Four sides
• Four corners
• Carbon in center
• Four bonds

• Four groups
• Two identical

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Left Side
Right Side
3
2
1
Bottom
Front
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5
4
(Looking at the surface of aconitase)
Which surface matches the active site of the
enzyme?
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Asymmetric Binding of Citrate
Left Face of Citrate
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Asymmetric labeling Of the TCA cycle by citrate
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Labeling in1st Turn
Acetyl CoA

a-KG
Citrate
Isocitrate
OAA
50
50
Succ CoA
OAA
Succinate

Fumarate
50
50
Malate
Label is randomized when malate is formed,
because of the two possible ways fumarate can
bind to fumarase.
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Labeling in 2nd Turn
25 of original lost as CO2
Another 25 of original lost as CO2

• 50 of original label lost in 2nd turn, all from
  1C of the original acetyl CoA
• 25 of original label lost in 3rd turn
• 12.5 of original label lost in 4th turn, etc.
• All of the carbons that were originally the
  carbonyl (green) of acetyl CoA have been
  liberated.

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Completing turn 2
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In the 3rd turn another 50 of remaining label is
lost
25 lost here
25 lost here
acetyl CoA oxaloacetate
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End