Download THE CITRIC ACID CYCLE

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BI25M1
THE CITRIC ACID
CYCLE
LECTURE 1:
AIMS:
To review:
the cycle in context,
by considering how and why the use
of oxygen to oxidise food molecules
evolved.
Lehninger:
Instant Notes:
Chapter 16
Section L1
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1 LIFE EVOLVED 3.5-4.0 x 109 YEARS
AGO.
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First humans
Extinction of dinosaurs
Origin of reptiles
Plants colonise land
500
Origin of multicellular organisms
1500
Oldest eukaryotic fossils
2500
Accumulation of atmospheric oxygen
from photosynthetic cyanobacteria
3500
Oldest prokaryotic fossils
4500
Origin of Earth
millions of years ago
Early organisms obtained energy by
taking up and breaking down simple
molecules present in the environment.
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They used this process:
molecule
simpler molecule
(higher
potential energy)
(lower
potential energy)
energy ‘released’
The breakdown of the simple ‘food’
molecule is an exergonic reaction,
(Energy Transformations lectures)
and the organisms were able to use the
‘released’ energy.
2 THE LATER PART OF GLYCOLYSIS
EVOLVED FIRST.
Among the early, simple food molecules
were ones similar to glyceraldehyde 3phosphate:
CHO
HCOH
CH2OP
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Some organisms managed to break this
down into the chemically simpler
pyruvate:
COOC=O
CH3
and ‘save’ some of the energy released as
ATP, or a similar molecule.
Thus evolved substrate-level
phosphorylation
(Energy Transformations lectures)
and the later part of what we now call
‘glycolysis’
(Carbohydrates
Lecture 4).
and
Intermediary
Metabolism
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3 EVOLUTION OF THE EARLY PART
OF GLYCOLYSIS
Simple food molecules were scarce. Some
organisms evolved ways of making them
from other, more complex molecules in
the environment. Chief among these were
hexoses, like glucose.
glucose
glyceraldehyde
3-phosphate
pyruvate
ATP
Thus evolved the early, sugar-splitting
(‘glyco-lysis’) part of glycolysis.
4 THE CHEMICAL NATURE OF
GLYCOLYSIS
Conversion of glucose
(or glyceraldehyde 3-phosphate)
to pyruvate is an oxidation.
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The oxidation centres on the glyceraldehyde
3-phosphate dehydrogenase -catalysed
reaction.
In the same reaction,
NAD+ is reduced to NADH + H+.
hexoses
glyceraldehyde
3-phosphate
NAD+
pyruvate
NADH + H+
Because of this,
early organisms using the ‘energyproviding’ pathway faced a problem:
when all the NAD+ is used up, flow stops.
To let flow continue, another step, which
regenerates NAD+, had to evolve.
Some organisms developed this step:
hexoses
glyceraldehyde
3-phosphate
NAD+
Others did this:
pyruvate
NADH + H+
NADH + H+
lactate
NAD+
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hexoses
glyceraldehyde
3-phosphate
NAD+
pyruvate
NADH + H+
ethanol
NADH + H+
NAD+
Both mechanisms survive today,
the former, for example,
in vigorously exercising muscle, and
when bacteria sour milk;
the latter, for example,
when yeast makes alcohol.
Unlike
hexoses
pyruvate,
NAD+
NADH + H+
which is a net oxidation,
hexoses
pyruvate
NAD+
is not.
NADH + H+
NADH + H+
lactate
or
ethanol
NAD+
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The latter processes are ‘fermentations’ defined as ‘energy-yielding’ pathways in
which no net change in the oxidation
state of the reactants occurs.
Because they involve no net oxidation,
fermentations harvest only a small part
of the potential energy of the glucose as
ATP.
(Just 2 mol ATP/mol glucose are generated
Carbohydrates and Intermediary Metabolism
Lecture 4).
In summary:
early organisms evolved ways of saving
some of the potential energy of molecules
in the environment using non-oxidative,
catabolic fermentations.
This involved making ATP by substratelevel phosphorylation.
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5 MEANWHILE, OTHER ORGANISMS
WERE USING SUNLIGHT.
Molecules like hexoses were scarce, and
organisms evolved several ways of
generating them for themselves.
All the ways worked like this:
sunlight
H2X
+
CO2
organic molecules
X
This is photosynthesis.
For some organisms, ‘H2X’
was (and for some still is)
H2S, or just H2.
Others,
including the cyanobacteria,
used, as ‘H2X’, a molecule very abundant
in many environments on Earth:
H2O.
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For such organisms, the released ‘X’ is,
of course, O2.
By about 2.5 x 109 years ago, their
activity led to accumulation of O2 in the
atmosphere to about present-day levels.
0
First humans
Extinction of dinosaurs
Origin of reptiles
Plants colonise land
500
Origin of multicellular organisms
1500
Oldest eukaryotic fossils
2500
Accumulation of atmospheric oxygen
from photosynthetic cyanobacteria
3500
Oldest prokaryotic fossils
4500
Origin of Earth
millions of years ago
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6 CONSEQUENCES OF OXYGEN
ACCUMULATION
Appearance in bulk of oxygen must have
caused many problems. Oxygen oxidises,
and disrupts the structure of the very
organic molecules of which organisms are
made.
Many organisms must have become
extinct.
Others survived by evolving antioxidant
defence mechanisms.
Some actually managed to exploit the
presence of the newly abundant molecule.
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7 USE OF OXYGEN TO OXIDISE
FOOD MOLECULES
Glucose (C6H12O6)
has many ‘high-energy’ electrons,
so-called because they are part of
electropositive H atoms.
When the electrons associate with
electronegative atoms, energy is released.
(Energy Transformations Lecture 3).
Some organisms evolved ways of
‘releasing’ and ‘saving’ this energy by
removing H atoms from food molecules,
and making the H atom electrons
associate with electronegative
NO3- or SO42-.
Some present-day micro-organisms still
do this.
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Other organisms evolved a different
method that was to become enormously
successful.
They made the H atom electrons
associate with atoms of one of the most
electronegative elements of all
- the very material that had become
abundant in their environment - oxygen.
Their method involved stripping H
atoms, two at a time, from what had
originally been a food molecule, like
glucose.
The H atom electrons were passed to the
oxidised form of a co-reactant, like NAD+
or FAD.
(The reduced co-reactant in some cases is then
used in an anabolic process useful to the organism
(Energy Transformations Lecture 3)).
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And the reduced co-reactant passed
the H atom electrons on,
through a series of redox carriers
(the ‘terminal respiratory system’),
until, eventually, they reached
deliberately taken into the cell,
and formed H2O.
O2
This accomplished complete breakdown
of the food molecule:
glucose +
6O2
6H2O + 6CO2
Unlike the earlier fermentations, it really
was an oxidation, and very exergonic.
Much more of the potential energy of the
food molecule was released, and could be
‘saved’ by making ATP.
Thus evolved oxidative phosphorylation
(Energy Transformations lectures).
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Because they could harness so much of
the potential energy of food molecules,
organisms using this oxygen-involving
process had an enormous evolutionary
advantage.
In addition,
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OXYGEN USE WIDENED THE
RANGE OF MOLECULES THAT
COULD BE EXPLOITED AS FOOD.
Some potential ‘foods’ have even more
‘high-energy’ electrons than hexoses,
for example, fatty acids, e.g.
CH3(CH2)14COO-.
[Such
molecules
resemble
petroleum
hydrocarbons. These are also fuels because of
their high content of H atom electrons
(Energy Transformations Lecture 3).]
The oxygen-involving apparatus that
evolved allowed complete oxidation of
hexoses like glucose (Section 7),
but it also developed in such a way as to
allow complete oxidation of fatty acids
(and amino-acids)
(Lipid Metabolism and How Organisms
Handle Nitrogen lectures).
So additional molecules became potential
foods.
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In summary:
the
primitive,
non-oxygen-involving
catabolism of hexoses survives, and is
important today.
For most cells, it is still the only way in
which ATP can be made without oxygen
(‘substrate-level phosphorylation’),
and is used in eukaryotic cells lacking
oxygen
and/or
the
oxygen-involving
‘apparatus’
(Energy Transformations Lecture 3).
[This is why glucose must be made
(by gluconeogenesis), if glycogen stores are depleted
and there is no dietary glucose
(Carbohydrates and Intermediary Metabolism Lecture
5).]
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9 WHAT IS THE OXYGENINVOLVING ‘APPARATUS’?
In eukaryotic cells, the complete
oxidation of food molecules occurs in
mitochondria.
(More primitive, partial catabolism of hexoses, by
glycolysis, occurs in the cytosol.)
H atoms are stripped from what were
originally food molecules, and their
electrons passed to NAD+ and FAD in a
process called the ‘citric acid cycle’.
Reduced NAD and FAD then pass the H
atom electrons through a series of redox
carriers (the ‘terminal respiratory
system’), until they reach O2.
Potential energy ‘released’ when they
reach O2 is ‘saved’ by making ATP
(‘oxidative phosphorylation’).
(Section 7; Energy Transformations Lecture 3).
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BI25M1
THE CITRIC ACID
CYCLE
LECTURE 2:
AIMS:
To review:
the functions and processes of the:
pyruvate dehydrogenase-catalysed
reaction;
citric acid cycle;
glyoxylate cycle.
Lehninger:
Instant Notes:
Chapter 16
Section L1
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10 HOW DID CELLS BEGIN
EXTRACTING FURTHER ENERGY
FROM FOOD USING OXYGEN?
Primitive, non-oxygen-involving
catabolism of hexoses (‘glycolysis’) works
like this (Sections 3,4):
ATP
hexose
glyceraldehyde
3-phosphate
NAD+
pyruvate
NADH + H+
NADH+ + H+
lactate
or ethanol
NAD+
The last step (to lactate or ethanol)
evolved to re-oxidise NADH made earlier
in the process (Section 4).
However, when O2 is being used, the last
step isn’t needed,
because the NADH can pass its H atom
electrons along the terminal respiratory
system to O2 (and so be re-oxidised).
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In summary:
some ATP has been made,
by substrate-level phosphorylation,
just as occurred in the absence of O2,
but,
additionally,
when the lactate/ethanol step is avoided,
the electrons from the two H atoms
stripped from the food molecule and
passed to NAD+, can now be passed
(eventually) to O2, and more ATP can be
made by oxidative phosphorylation.
So, just avoiding the final step of the
primitive process has begun the business
of harvesting extra energy.
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11 IF PYRUVATE ISN’T REDUCED
TO LACTATE OR ETHANOL,
WHAT HAPPENS TO IT?
(Carbohydrates and Intermediary Metabolism Lecture 4)
It is further oxidised.
Another pair of ‘high-energy’-electroncontaining H atoms are stripped away,
Again, their electrons are passed to
NAD+.
And, again, the electrons can pass to O2,
and more ATP can be made by oxidative
phosphorylation.
The oxidation of pyruvate:
is catalysed by pyruvate dehydrogenase;
is complex;
is essentially irreversible under intracellular
conditions;
occurs in mitochondria in eukaryotic cells.
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This is what happens:
Coenzyme A
CO2
pyruvate
COOC=O
CH3
acetyl CoA
NAD+
NADH + H+
CH3CSCoA
O
[Coenzyme A is a nucleotide-containing molecule that has an –SH group
that can form a thio-ester link with carboxylic acids. (Lehninger, Edition 4
p.603; Edition 5 p.617]
In summary:
Further potential energy of the food
molecule is ‘released’ and ‘saved’, as two
more H atoms are stripped off and used
to
make
ATP
by
oxidative
phosphorylation.
Notice the progressive break-down:
glucose
pyruvate
6C
3C
acetyl CoA
2C
[And remember
glucose +
6O2
6H2O
+
6CO2?
This is the point where 2 of those 6 CO2 are made.]
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12 WHAT HAPPENS TO THE ACETYL
CoA?
It is oxidised to CO2.
The remaining H atoms are stripped off,
two at a time.
The electrons of most are passed to
NAD+;
some are passed to FAD.
The
oxidation of acetyl CoA;
stripping away of the H atoms;
reduction of NAD+ and FAD;
all occur by a mechanism called
the citric acid cycle
(or tricarboxylic acid, TCA or Krebs’
cycle),
which, in eukaryotic cells, occurs in
mitochondria.
Lehninger, Edition 4 p.607; Edition 5 p.621
Instant Notes, p.369
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We can think of the cycle as a ‘black box’
(physicists talk about such things):
it has to be there for the process to occur,
but is itself unchanged during the process.
Thus,
flow through the cycle causes
no net gain or loss of any of the 9
intermediates.
The net changes that DO occur
when one acetyl CoA is oxidised
(i.e during one turn of the cycle)
are:
acetyl CoA
2CO2
3 (NADH + H+)
3H2O
1 FADH2
1 GTP
(this is made from GDP,
and is energetically
equivalent to ATP)
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A slightly more complicated version:
acetyl CoA
2C
oxaloacetate
4C
6C
4C
6C
4C
6C
citrate
NADH + H+
NADH + H+ CO2
FADH2
succinate
4C
4C
5C
-ketoglutarate
NADH + H+
CO2
GTP
Yet another way of thinking about the
cycle is:
Attachment to carrier
Carrier
regenerated
Breaking
carrier
where the ‘carrier’ is oxaloacetate.
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The cycle is part of an oxygen-involving
‘apparatus’ concerned not just with
complete oxidation of glucose,
but also of
fatty acids and
amino-acids
(Section 8).
glucose
fatty acids
pyruvate
 oxidation
acetyl CoA
amino-acids
CO2
(Lipid Metabolism and How Organisms Handle
Nitrogen lectures).
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13 THE CYCLE, AS A CYCLE, IS
PURELY CATABOLIC.
C flow through the cycle is:
2 C in (as acetyl group of acetyl CoA)
2 C out (as CO2),
(Section 12)
with no net production (or loss) of any of
the 9 intermediates for possible use in
anabolism.
To emphasise this lack of an anabolic
function, we can consider:
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14 CARBOHYDRATE-FATTY ACID
INTER-CONVERSION
We know that:
glucose
pyruvate
glycolysis
acetyl CoA
pyruvate
dehydrogenase
and that acetyl CoA can be catabolised
through the citric acid cycle (Section 12).
Acetyl CoA is also the building block of
fatty acids (Lipid Metabolism lectures),
so this process can also occur:
glucose
pyruvate
acetyl CoA
fatty acids
fats
Though this route, excess dietary
carbohydrate is converted to fat and
stored in adipose tissue.
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15 IF GLUCOSE CAN BE CONVERTED
TO FATTY ACIDS, IS THE REVERSE
TRUE?
How about this route?
glucose
pyruvate
acetyl CoA
fatty acids
Well, it’s not possible,
because the pyruvate-acetyl CoA reaction
is irreversible (Section 11).
How about this?
Oxaloacetate
is
part
of
the
gluconeogenesis pathway (Carbohydrates
and Intermediary Metabolism Lecture 5)
and ALSO one of the 9 citric acid cycle
intermediates (Section 12).
And fatty acids are catabolised to acetyl
CoA (Lipid Metabolism lectures),
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so could the fatty acids be converted into
glucose like this?
fatty acid
acetyl CoA
oxaloacetate
glucose
The answer is, again, no.
There is no net flow of C from fatty acid
to glucose by this route, because there is
no net synthesis through the cycle of
oxaloacetate (or any of the other cycle
intermediates).
The function of the cycle, remember,
isn’t to make cycle intermediates, it’s to
catabolise acetyl CoA to CO2 (Section 13).
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Another way of looking at this is to
remember what oxaloacetate is doing:
it accepts the 2 C from acetyl CoA into
the cycle;
if it’s doing that, it can’t simultaneously
be tapped off to make glucose.
In summary:
in many organisms
(including all animal cells),
fatty acids and acetyl CoA are not
precursors of hexoses.
(They were NOT among the short list of
gluconeogenic precursors in Carbohydrates and
Intermediary Metabolism Lecture 5).
And the cycle, as a cycle, is purely
catabolic.
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Having said that
parts of the cycle can form sections of
linear metabolic pathways used in
anabolism.
For example,
some amino-acids are metabolised to
-ketoglutarate (one of the 9 intermediates),
and through the route below,
act as a source of glucose.
glucose
oxaloacetate
some
amino-acids
-ketoglutarate
part of the
citric acid cycle
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What happens is that the amino-acids
‘feed-in’ C, which replenishes oxaloacetate
tapped off to make glucose, and part of the
cycle is involved in this anabolic flow.
Similar feed-in reactions enable other
intermediates to be tapped off for
anabolism.
These ‘feed-in’ reactions are called
anaplerotic (or replenishing) reactions.
[How Organisms Handle Nitrogen Lecture 4]
Because of this anabolic activity, the
citric acid cycle is sometimes said to be
amphibolic
(i.e. to have catabolic and anabolic
activity).
But remember,
as a cycle, it is purely catabolic.
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16 THE GLYOXYLATE CYCLE
This is evolution’s answer to the problem
of
converting
fatty
acids
into
carbohydrates.
Some plant seeds store fat.
At germination, the plant needs
carbohydrates for many purposes,
including building its structure.
It lacks leaves, and so can’t make
carbohydrates by photosynthesis.
It needs to convert fatty acids from its fat
stores into glucose.
Similarly, some bacteria,
protozoa and
fungi
grow on fatty acids with little or no
carbohydrate present.
They, too, must convert fatty acids into
glucose.
Such organisms have 2 enzymes, which
animal cells lack.
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These work with enzymes of part of the
citric acid cycle, to produce another,
anabolic, cycle:
the glyoxylate cycle.
C flow through the citric acid cycle is:
(Section 12)
2Cin
acetyl CoA
2 CO2
2Cout
C flow through the glyoxylate cycle is:
fatty acids
2Cin
acetyl CoA
part of the
citric acid cycle
*the 2 additional enzymes
malate
4C
isocitrate
6C
*
*
glyoxylate
2C
acetyl CoA
2Cin
succinate
4Cout
carbohydrates
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or, more simply:
2Cin
acetyl CoA
succinate
4Cout
acetyl CoA
2Cin
In plant seeds, the two additional
enzymes are in organelles called
glyoxysomes.