Tricarboxylic Acid Cycle

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Tricarboxylic Acid Cycle TCA Cycle
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Learning Objectives
Write out the TCA cycle starting with pyruvate
and include the coenzymes involved in each
reaction step clearly identify the irreversible
reactions.
Describe the TCA cycle where are the enzymes
located, the net result of one turn of the cycle,
why the cycle is important in terms of eventual
ATP production.
Identify the only TCA cycle enzyme that is
membrane bound.
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Under aerobic conditions, glycolysis is just the
first stage in the complete oxidation of glucose
to CO2 and H2O.
The pyruvate produced by glycolysis and other
organic fuel molecules are oxidized to yield
two-carbon fragments in the form of
acetyl-coenzyme A (acetyl-CoA).
The acetyl groups are fed into the TCA cycle
where they are enzymatically oxidized to CO2.
The energy released is conserved in the reduced
electron carriers NADH and FADH2.
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Rough endoplasmic reticulum
Structure of a mitochondria
The enzymes of the TCA cycle are found in the
mitochondria.
The extensive infolding of the inner membrane
(cristae) increases the surface area of this
membrane.
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Conversion of pyruvate to acetyl-CoA
The reaction catalyzed by pyruvate dehydrogenase
is an oxidative decarboxylation an irreversible
oxidation process in which the carboxyl group of
pyruvate is removed as CO2, and the two remaining
carbons become the acetyl group of acetyl-CoA.
There can be no synthesis of glucose starting
from acetyl-CoA.
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The pyruvate dehydrogenase complex (three
enzymes E1, E2 and E3) requires 5 coenzymes or
prosthetic groups thiamine pyrophosphate (TPP)
thiamine flavin adenine dinucleotide (FAD)
riboflavin coenzyme A (CoA) pantothenate nico
tine adenine dinucleotide (NAD) niacin lipoate
essential vitamin required in human nutrition
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Coenzyme form of vitamine B1
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FADH
FADH2
fully reduced
(semiquinone)
(quinone)
Flavin adenine dinucleotide (FAD)
(oxidized)
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Coenzyme A
The thioester linkage in acetyl-CoA is a
high-energy bond.
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NADH (reduced)
NAD (oxidized)
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Lipoic acid (lipoate) in amide linkage with the
side chain of a lysine residue
Polypeptide chain of E2 (dihydrolipoyl
transacetalase)
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Reaction cycle of pyruvate dehydrogenase complex
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Reaction cycle of pyruvate dehydrogenase
1 Pyruvate reacts with bound TPP on E1, and
undergoes decarboxylation to the hydroxyethyl
derivative. 2 Two electrons and the acetyl
group are transferred from TPP to the
lipoyllysine of the core enzyme E2. 3 This
step is a transesterification in which SH group
of CoA replaces the SH group of E2 to yield
acetyl-CoA and the fully reduced dithiol form of
the lipoyl group. 4 E3 (dihydrolipoyl
dehydrogenase) promotes transfer of two hydrogen
atoms (including electrons) from the reduced
lipoyl groups of E2 to the FAD prosthetic group
of E3. This restores the oxidized form of the
lipoyl group of E3. 5 The reduced FADH2 of E3
transfers a hydride ion (H-) to NAD, forming
NADH H. The enzyme complex is now ready for
another catalytic cycle.
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acetyl-CoA
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citrate
oxaloacetate
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TCA Cycle
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malate
cis-aconitate
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fumarate
isocitrate
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succinate
a-ketoglutarate
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4
succinyl-CoA
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1 Formation of citrate
The first reaction in the cycle, catalyzed by
citrate synthase, is the condensation of
acetyl-CoA with oxaloacetate to form citrate.
Citroyl-CoA is a transient intermediate formed
on the active site of the enzyme. It rapidly
undergoes hydrolysis to free CoA and citrate.
The hydrolysis of this high-energy thioester
intermediate make the forward reaction highly
exergonic (release of free energy DG is
negative)
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(open form)
Citrate synthase
binding of oxaloacetate (yellow) and
carboxymethyl-CoA (red)
Citrate synthase undergoes a large conformational
change upon binding of substrates.
(closed form)
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2 Formation of isocitrate via cis-aconitate
The enzyme aconitase catalyzes the reversible
transformation of citrate to isocitrate through
the intermediary formation of the tricarboxylic
acid cis-aconitate, which normally does not
dissociate from the enzyme active site.
Aconitase can promote the addition of H2O to the
double bond of cis-aconitate in two different
ways, one leading to citrate and the other
leading to isocitrate.
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The active site of aconitase contains an
iron-sulfur center or cluster which acts in the
binding of substrate and the catalytic addition
or removal of H2O.
Iron-sulfur cluster (red) with bound citrate
(blue). Three cysteine residues bind three iron
atoms the fourth iron is bound to one of the
carboxyl groups of citrate. B is a basic amino
acid residue (arginine?) that helps position
citrate in the active site.
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3 Oxidation of isocitrate to a-ketoglutarate
and CO2
There are two forms of this enzyme a NAD
dependent enzyme found in the mitochondrial
matrix and a NADP dependent enzyme found in
both the mitrochondria and the cytosol. The
cytosolic enzyme may function to supply NADPH for
reductive anabolic reactions.
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4 Oxidation of a-ketoglutarate to succinyl-CoA
and CO2
This is another oxidative decarboxylation step
with an enzyme complex very similar to pyruvate
dehydrogenase. This complex also has three
activities (E1, E2, and E3) with enzyme bound TPP
and lipoyl moieties.
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5 Conversion of succinyl-CoA to succinate
Animal cells have two isozymes of succinyl-CoA
synthase one specific for GTP, and another
specific for ATP.
Succinyl-CoA, like acetyl-CoA, has a high-energy
thioester bond. The energy release in the
breakage of this bond is used to drive the
formation of GTP (or ATP) with the concomitant
formation of succinate. This is another
substrate-phosphorylation step like the
ATP-forming reaction in glycolysis.
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The succinyl-CoA synthase reaction
A phosphoryl group (from inorganic phosphate)
replaces CoA in the succinyl-CoA bound to the
enzyme, forming a high-energy acyl phosphate.
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In the second step, the succinyl phosphate
donates its phosphoryl group to a histidine (His)
residue on the enzyme. This forms a high-energy
phosphohistidyl enzyme.
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In the last step of the reaction sequence, the
phosphoryl group is transferred to GDP (or ADP)
to produce GTP (or ATP).
All cells contain an enzyme (nucleoside
diphosphate kinase) to transfer phosphoryl groups
between a nucleoside triphosphate and a
nucleotide diphosphate.
Mg2
GTP ADP GDP ATP
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Succinyl-CoA synthase from E. coli
Coenzyme A
(red stick structure)
The active site contains part of the a (blue) and
b (brown) subunits.
Two critical helices, one from each subunit (dark
blue and dark brown), are orientated to stabilize
the phosphohistidyl group (orange) on the enzyme.
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6 Oxidation of succinate to fumarate
Succinate is oxidized to fumarate by the
flavoprotein, succinate dehydrogenase. This is
the only TCA cycle enzyme that is membrane-bound.
In eukaryotes, it is tightly attached to the
inner mitochondrial membrane. The beef heart
enzyme contains three different iron-sulfur
clusters and a covalently attached FAD. The
membrane location of succinate dehydrogenase
allows electrons to pass from succinate through
FADH2 and the iron-sulfur clusters, and then
enter the chain of electron carriers in the
mitochondrial inner membrane.
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7 Hydration of fumarate to malate
Fumarase is highly stereospecific. It catalyzes
hydration of the trans double bond of fumarate,
but not the cis double bond of maleate (the cis
isomer of fumarate). In the reverse direction,
D-malate is not a substrate.
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8 Oxidation of malate to oxaloacetate
The equilibrium of this reaction lies far to the
left under standard thermodynamic conditions, but
in the intact cell, oxaloacetate is continually
removed by the highly exergonic citrate synthase
1. This keeps the concentration of oxaloacetate
in the cell extremely low (lt 10-6 M), and pulls
the malate dehydrogenase reaction toward the
formation of oxaloacetate.
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A two-carbon acetyl group entered the cycle.
One turn of the TCA cycle
At the end of the cycle, one molecule of
oxaloacetate is regenerated.
Two carbons emerged from the cycle as CO2
The energy released by these oxidations is
conserved in the reduction of 3 NAD and 1 FAD,
and the production of one GTP (or ATP).
The two carbons appearing as CO2 are not the same
two in the acetyl-CoA that entered the cycle.
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Although the TCA cycle itself generates only one
ATP per turn, the four oxidation steps in the
cycle provide a large flow of electrons into the
respiratory chain (electron transfer chain) via
NADH and FADH2. This leads to the formation of a
large number of ATP molecules during oxidative
phosphorylation.
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(No Transcript)
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Why is the oxidation of acetate so complicated?
The role of the TCA cycle is not confined to the
oxidation of acetate. This pathway is the hub of
intermediary metabolism. Four- and five-carbon
end products of many catabolic process feed into
the TCA cycle to serve as fuels. Oxaloacetate is
produced from the degradation of aspartic acid
a-ketoglutarate is produced from glutamic acid
when proteins are degraded. TCA cycle
intermediates are also used in anabolic pathways.
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Role of the TCA cycle in anabolism
Anaplerotic reactions red arrows
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As intermediates of the TCA cycle are removed to
serve as biosynthetic precursors, they are
replenished by anaplerotic reactions.
anaplerotic reaction an enzyme-catalyzed
reaction that can replenish the supply of
intermediates in the TCA cycle.
Under normal circumstances, the reactions by
which cycle intermediates are siphoned off into
other pathways and those by which they are
replenished are in dynamic balance. Thus,
concentrations of TCA cycle intermediates remain
almost constant.
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Under normal conditions, the rates of glycolysis
and of the TCA cycle are integrated so that only
as much glucose is metabolized to pyruvate as is
needed to supply the TCA cycle with fuel. The
rate of glycolysis is matched to the rate of the
TCA cycle through its inhibition by high levels
of ATP and NADH. Citrate, the product of the
first step in the TCA cycle, is an important
allosteric inhibitor of phosphofructokinase-1 in
the glycolytic pathway.
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Regulation of phosphofructokinase-1
High citrate concentrations increase the
inhibitory effect of ATP.
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Thiamine deficiency
Thiamine-deficient animals (and humans) are
unable to oxidize pyruvate normally. This is
particularly important for the brain which
obtains all its energy from the aerobic oxidation
of glucose in a pathway that necessarily includes
pyruvate dehydrogenase. Beriberi, a disease that
results from thiamine deficiency, is
characterized by loss of neural function. This
disease occurs primarily in populations that rely
on a diet consisting mainly of white (polished)
rice. (The hulls of the rice have been removed
in which most of the thiamine is found).
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Regulation of the TCA Cycle
Activation
Inhibition