Download The Citric Acid Cycle

The Citric Acid Cycle
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The aerobic processing of glucose
start with the complete oxidation of
glucose derivatives to CO2.
This oxidation takes place in the citric
acid cycle, a series of reactions also
known as the tricarboxylic acid (TCA)
cycle or the Krebs cycle.
The citric acid cycle is the final common
pathway for the oxidation of fuel molecules.
It also serves as a source of building
blocks for biosyntheses.
Most fuel molecules enter the cycle as
acetyl CoA. The link between glycolysis and
the citric acid cycle is the oxidative
decarboxylation of pyruvate to form acetyl
CoA.
In eukaryotes, this reaction and those of
the cycle take place inside mitochondria, in
contrast with glycolysis, which takes place in
the cytosol.
Biosynthetic Roles of the Citric Acid Cycle. Intermediates drawn off for
biosyntheses (shown by red arrows) are replenished by the formation of
oxaloacetate from pyruvate.
Mitochondrion.
The double membrane of the mitochondrion is
evident in this electron micrograph. The numerous
invaginations of the inner mitochondrial membrane are called
cristae. The oxidative decarboxylation of pyruvate and the
sequence of reactions in the citric acid cycle take place within
the matrix.
Pyruvate Transport
(Antiporter)
Pyruvate
Pyruvate Carrier
OH
-
Overview of the Citric Acid Cycle.
The citric acid
cycle oxidizes twocarbon units,
producing two
molecules of CO2,
one molecule of
GTP, and highenergy electrons in
the form of NADH
and FADH2.
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The function of the citric acid cycle is the
harvesting of high energy electrons from
carbon fuels.
Note that the citric acid cycle itself neither
generate large amount of ATP nor includes
oxygen as a reactant. Instead, it removes
electrons from acetyl CoA and uses these
electrons to form NADH and FADH2.
The formation of Acetyl Coenzyme A from
Pyruvate.
Pyruvate is oxidatively
decarboxylated by the
pyruvate dehydrogenase
complex to form acetyl CoA.
This irreversible reaction is
the link between glycolysis
and citric acid cycle.
Pyruvate dehydrogenase complex of E. coli
Enzyme
Abbreviation
Number of
chains
Prosthetic
group
Reaction catalyzed
Pyruvate
dehydrogenase
component
E1
24
TPP
Oxidative
decarboxylation
of pyruvate
Dihydrolipoyl
transacetylase
E2
24
Lipoamide
Transfer of the
acetyl group to
CoA
Dihydrolipoyl
dehydrogenase
E3
12
FAD
Regeneration of
the oxidized
form of
lipoamide
The Formation of Acetyl Coenzyme A
from Pyruvate
The reaction requires the participation of the
three enzymes of the pyruvate dehydrogenase
complex, each composed of several polypeptide
chains, and five coenzymes:
–
–
–
–
–
B1
thiamine pyrophosphate (TPP),
lipoic acid,
FAD,
CoA
NAD+.
Acetyl Coenzyme A
(Acetyl CoA).
B5
The conversion of pyruvate into acetyl
CoA consists of three steps:
123-
Decarboxylation,
Oxidation,
Transfer of the acetyl group to CoA.
Thiazole ring
Carbanion
Mechanism of the Decarboxylation Reaction of E1 ,
The Pyruvate Dehydrogenase Component of the
Pyruvate Dehydrogenese Complex.
The hydroxyethyl group attached to TPP is oxidized to form
an acetyl group and concomitantly transferred to
lipoamide, a derivative of lipoic acid that is linked to the
side chain of a lysine residue by an amide linkage.
The oxidant in this reaction is the disulfide group of
lipoamide, which is reduced to its disulfhydryl form. This
reaction, also catalyzed by the pyruvate dehydrogenase
component E1, yields acetyllipoamide.
Third, the acetyl group is transferred
from acetyllipoamide to CoA to form
acetyl CoA.
Dihydrolipoyl transacetylase (E2)
catalyzes this reaction.
The pyruvate dehydrogenase complex cannot
complete another catalytic cycle until the
dihydrolipoamide is oxidized to lipoamide.
In a fourth step, the oxidized form of
lipoamide is regenerated by dihydrolipoyl
dehydrogenase (E3).
Two electrons are transferred to an FAD
prosthetic group of the enzyme and then to
NAD+.
Citrate Synthase Forms Citrate from
Oxaloacetate and Acetyl Coenzyme A
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The citric acid cycle begins with the condensation of a fourcarbon unit, oxaloacetate, and a two-carbon unit, the acetyl
group of acetyl CoA.
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Oxaloacetate reacts with acetyl CoA and H2O to yield citrate
and CoA.
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This reaction, which is an aldol condensation followed by a
hydrolysis, is catalyzed by citrate synthase.
Citrate is isomerized into isocitrate to enable the sixcarbon unit to undergo oxidative decarboxylation.
The isomerization of citrate is accomplished by a
dehydration step followed by a hydration step.
The result is an interchange of a hydrogen atom and a
hydroxyl group. The enzyme catalyzing both steps is called
aconitase because cis-aconitate is an intermediate.
Isocitrate Is Oxidized and
Decarboxylated to α-Ketoglutarate
The oxidative decarboxylation of isocitrate is catalyzed
by isocitrate dehydrogenase.
The intermediate in this reaction is oxalosuccinate, an
unstable β-ketoacid. While bound to the enzyme, it loses CO2
to form α-ketoglutarate.
Succinyl Coenzyme A Is Formed by the
Oxidative Decarboxylation of α-Ketoglutarate
α-Ketoglutarate dehydrogenase
A High Phosphoryl-Transfer Potential Compound Is
Generated from Succinyl Coenzyme A
The cleavage of the thioester bond of succinyl CoA is
coupled to the phosphorylation of a purine nucleoside
diphosphate, usually GDP.
This reaction is catalyzed by succinyl CoA synthetase
(succinate thiokinase).
Oxaloacetate Is Regenerated by the
Oxidation of Succinate
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The final stage of the citric acid cycle.
A methylene group is converted into a carbonyl group in three
steps;
– Oxidation (Succinate dehydrogenase)
– Hydration (Fumarase)
– Second oxidation (Malate dehydrogenase)
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Not only is oxaloacetate thereby regenerated for another round
of the cycle, but also more energy is extracted in the form of
FADH2 and NADH.
Stoichiometry of Citric
Acid Cycle.
Acetyl CoA + 3NAD + FAD + GDP + Pi + 2H2O
2 CO2 + 3NADH + FADH2 + GTP + 2H+ +CoA
Reactions of the Pyruvate Dehydrogenase Complex. At the top (center), the
enzyme (represented by a yellow, a blue, and two red spheres) is
unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated
to form the hydroxyethyl TPP. (2) The dihydrolipoyl arm of E2 moves into
the active site of E1. (3) E1 catalyzes the transfer of the two-carbon group
to the dihydrolipoyl group to form the acetyl-lipoyl complex. (4) E2
catalyzes the transfer of the acetyl moiety to CoA to form the product
acetyl CoA. The disulfhydryl lipoyl arm then swings to the active site of E3.
E3 catalyzes (5) the reduction of the lipoic acid and (6) the transfer of the
protons and electrons to NAD+ to complete the reaction cycle.
1.
Pyruvate is decarboxylated at the active site of E1, forming the substituted TPP
intermediate, and CO2 leaves as the first product. This active site lies within the
E1 complex, connected to the enzyme surface by a 20-Å-long hydrophobic
channel.
2.
E2 inserts the lipoyl-lysine arm of the lipoamide domain into the channel in E1.
3.
E1 catalyzes the transfer of the acetyl group to the lipoamide. The acetylated
lipoyl-lysine arm then leaves E1 and enters the E2 cube through 30 Å windows
on the sides of the cube to visit the active site of E2, located deep in the cube
at the subunit interface.
4.
The acetyl moiety is then transferred to CoA, and the second product, acetyl
CoA, leaves the cube. The reduced lipoyl-lysine arm then swings to the active
site of the E3 flavoprotein.
5.
At the E3 active site, the lipoamide acid is oxidized by coenzyme FAD.
6.
The final product, NADH, is produced with the reoxidation of FADH2, and the
reactivated lipoamide is ready to begin another reaction cycle.
The structural integration of three kinds of enzymes makes the coordinated
catalysis of a complex reaction possible. The proximity of one enzyme to
another increases the overall reaction rate and minimizes side reactions. All the
intermediates in the oxidative decarboxylation of pyruvate are tightly bound to
the complex and are readily transferred because of the ability of the lipoyllysine arm of E2 to call on each active site in turn.
Step
Reaction
Prosthetic group
Type*
Aconitase
Fe-S
b
Aconitase
Fe-S
c
2a
Citrate
2b
cis-Aconitate+ H2O
3
Isocitrate + NAD+
4
α-Ketoglutarate + NAD+ + CoA
NADH
5
Succinyl CoA + Pi + GDP
6
Succinate + FAD (enzyme-bound)
(enzyme-bound)
7
Fumarate + H2O
l-malate
Furmarase
c
8
l-Malate + NAD+
oxaloacetate + NADH + H+
Malate dehydrogenase
e
*
cis-aconitate + H2O
Enzyme
isocitrate
α-ketoglutarate + CO2 + NADH
succinyl CoA + CO2 +
succinate + GTP + CoA
fumarate + FADH2
Isocitrate dehydrogenase
α-Ketoglutarate dehydrogenase
complex
d+e
Lipoic acid, FAD, TPP
Succinyl CoA synthetase
Succinate dehydrogenase
d+e
f
FAD, Fe-S
e
Reaction type: (a) condensation; (b) dehydration; (c) hydration; (d) decarboxylation; (e) oxidation; (f) substrate-level
phosphorylation.
1. Two carbon atoms enter the cycle in the condensation of an
acetyl unit (from acetyl CoA) with oxaloacetate. Two carbon
atoms leave the cycle in the form of CO2 in the successive
decarboxylations catalyzed by isocitrate dehydrogenase and αketoglutarate dehydrogenase. Interestingly, the results of
isotope-labeling studies revealed that the two carbon atoms that
enter each cycle are not the ones that leave.
2. Four pairs of hydrogen atoms leave the cycle in four oxidation
reactions. Two molecules of NAD+ are reduced in the oxidative
decarboxylations of isocitrate and α-ketoglutarate, one molecule
of FAD is reduced in the oxidation of succinate, and one
molecule of NAD+ is reduced in the oxidation of malate.
3. One compound with high phosphoryl transfer potential,
usually GTP, is generated from the cleavage of the thioester
linkage in succinyl CoA.
4. Two molecules of water are consumed: one in the synthesis
of citrate by the hydrolysis of citryl CoA and the other in the
hydration of fumarate.
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Glycolysis has both an aerobic and an
anaerobic mode, whereas the citric acid cycle
is strictly aerobic.
Glycolysis can proceed under anaerobic
conditions because NAD+ is regenerated in
the conversion of pyruvate into lactate.
Control of the Citric Acid Cycle
ƒThe citric acid cycle is regulated
primarily by the concentration of ATP
and NADH.
ƒThe key control points are the
enzymes:
isocitrate dehydrogenase
and
α-ketoglutarate dehydrogenase.
Oxidative Phosphorylation
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The NADH and FADH2 formed in glycolysis, fatty acid
oxidation, and the citric acid cycle are energy-rich
molecules because each contains a pair of electrons
having a high transfer potential.
When these electrons are used to reduce molecular
oxygen to water, a large amount of free energy is
liberated, which can be used to generate ATP.
Oxidative phosphorylation is the process in which ATP
is formed as a result of the transfer of electrons from
NADH or FADH2to O2 by a series of electron carriers
carriers..
This process, which takes place in mitochondria, is the
major source of ATP in aerobic organisms
For example, oxidative phosphorylation generates 26
of the 30 molecules of ATP that are formed when
glucose is completely oxidized to CO2 and H2O.
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The flow of electrons from NADH or FADH2 to O2 through
protein complexes located in the mitochondrial inner
membrane leads to the pumping of protons out of the
mitochondrial matrix.
The resulting uneven distribution of protons generates a
pH gradient and a transmembrane electrical potential that
creates a proton-motive force.
ATP is synthesized when protons flow back to the
mitochondrial matrix through an enzyme complex.
Thus, the oxidation of fuels and the phosphorylation of
ADP are coupled by a proton gradient across the inner
mitochondrial membrane.
Sequence of Electron Carriers in the Respiratory Chain.
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Oxidative phosphorylation is the culmination
of a series of energy transformations that are
called cellular respiration or simply respiration
in their entirety.
First, carbon fuels are oxidized in the citric
acid cycle to yield electrons with high transfer
potential. Then, this electron-motive force is
converted into a proton-motive force and,
finally, the proton-motive force is converted
into phosphoryl transfer potential.
The conversion of electron-motive force into
proton-motive force is carried out by three
electron-driven proton pumps:
–
–
–
–
NADH-Q oxidoreductase,
Q-cytochrome c oxidoreductase,
and cytochrome c oxidase.
These large transmembrane complexes contain
multiple oxidation-reduction centers, including
quinones, flavins, iron-sulfur clusters, hemes, and
copper ions.
Complex I
Complex II
FADH2
Complex III
Complex IV
Does not
pump protons
Cyt C (reduced)
Cyt C (oxidized)
4H+
Fe
O2
4H+ (Pumped protons)
Cu
2 H2 O
Four “chemical” protons are
Taken up from the matrix
side to reduce one molecule
of O2 to two molecules of
H2O. Four additional
“pumped” protons are
transported out of the matrix
and released on the cytosolic
side in the course of the
reaction. The pumped
protons double the efficiency
of free-energy storage in the
form of a proton gradient.
4H+ (chemical protons)
A Proton Gradient Powers the
Synthesis of ATP
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The final phase of oxidative phosphorylation is
carried out by ATP synthase, an ATP-synthesizing
assembly that is driven by the flow of protons back
into the mitochondrial matrix.
Components of this remarkable enzyme rotate as
part of its catalytic mechanism.
Oxidative phosphorylation vividly shows that proton
gradients are an interconvertible currency of free
energy in biological systems.
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In oxidative phosphorylation, the synthesis
of ATP is coupled to the flow of electrons
from NADH or FADH2 to O2 by a proton
gradient across the inner mitochondrial
membrane. Electron flow through three
asymmetrically oriented transmembrane
complexes results in the pumping of protons
out of the mitochondrial matrix and the
generation of a membrane potential. ATP is
synthesized when protons flow back to the
matrix through a channel in an ATPsynthesizing complex, called ATP synthase
(also known as F0F1-ATPase). Oxidative
phosphorylation exemplifies a fundamental
theme of bioenergetics: the transmission of
free energy by proton gradients.
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The flow of electrons through Complexes I, III, and IV leads
to the transfer of protons from the matrix side to the
cytosolic side of the inner mitochondrial membrane. A
proton-motive force consisting of a pH gradient (matrix side
basic) and a membrane potential (matrix side negative) is
generated. The flow of protons back to the matrix side
through ATP synthase drives ATP synthesis. The enzyme
complex is a molecular motor made of two operational units:
a rotating component and a stationary component. The
rotation of the g subunit induces structural changes in the b
subunit that result in the synthesis and release of ATP from
the enzyme. Proton influx provides the force for the rotation.
The flow of two electrons through NADH-Q oxidoreductase,
Q-cytochrome c oxidoreductase, and cytochrome c oxidase
generates a gradient sufficient to synthesize 1, 0.5, and 1
molecule of ATP, respectively. Hence, 2.5 molecules of ATP
are formed per molecule of NADH oxidized in the
mitochondrial matrix, whereas only 1.5 molecules of ATP are
made per molecule of FADH2 oxidized because its electrons
enter the chain at QH2, after the first proton-pumping site.
about 30 molecules of ATP are formed
when glucose is completely oxidized to
CO2; this value supersedes the
traditional estimate of 36 molecules of
ATP. Most of the ATP, 26 of 30
molecules formed, is generated by
oxidative phosphorylation. Recall that
the anaerobic metabolism of glucose
yields only 2 molecules of ATP.
Sites of Action of Some Inhibitors
of Electron Transport.
Uncoupling of Oxidative Phosphorylation.
2,4 –Dinitrophenol, a lipid-soluble substance, can carry
protons across the inner mitochondrial membrane.
Figure 18.45. Action of an Uncoupling Protein. Uncoupling protein-1
(UCP-1) generates heat by permitting the influx of protons into the
mitochondria without the synthesis of ATP.
Regulated Uncoupling Leads to the Generation of Heat
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The uncoupling of oxidative phosphorylation is a means of
generating heat to maintain body temperature in hibernating
animals, in some newborn animals (including human beings),
and in mammals adapted to cold. Brown adipose tissue, which
is very rich in mitochondria (often referred to as brown fat
mitochondria), is specialized for this process of nonshivering
thermogenesis. The inner mitochondrial membrane of these
mitochondria contains a large amount of uncoupling protein
(UCP), here UCP-1, or thermogenin, a dimer of 33-kd subunits
that resembles ATP-ADP translocase. UCP-1 forms a pathway
for the flow of protons from the cytosol to the matrix.
In essence, UCP-1 generates heat by short-circuiting the
mitochondrial proton battery. This dissipative proton pathway
is activated by free fatty acids liberated from triacylglycerols
in response to hormonal signals, such as b-adrenergic
agonists .
Many Shuttles Allow Movement
Across Mitochondrial Membranes.
Electrons from Cytosolic NADH Enter Mitochondria
by Shuttles.
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Recall that the glycolytic pathway generates NADH in the cytosol in
the oxidation of glyceraldehyde 3-phosphate, and NAD+ must be
regenerated for glycolysis to continue.
How is cytosolic NADH reoxidized under aerobic conditions? NADH
cannot simply pass into mitochondria for oxidation by the respiratory
chain, because the inner mitochondrial membrane is impermeable to
NADH and NAD+. The solution is that electrons from NADH, rather
than NADH itself, are carried across the mitochondrial membrane.
One of several means of introducing electrons from NADH into the
electron transport chain is the glycerol 3-phosphate shuttle.
The first step in this shuttle is the transfer of a pair of electrons from
NADH to dihydroxyacetone phosphate, a glycolytic intermediate, to
form glycerol 3-phosphate.This reaction is catalyzed by a glycerol 3phosphate dehydrogenase in the cytosol. Glycerol 3-phosphate is
reoxidized to dihydroxyacetone phosphate on the outer surface of
the inner mitochondrial membrane by a membrane-bound isozyme of
glycerol 3-phosphate dehydrogenase. An electron pair from glycerol
3-phosphate is transferred to a FAD prosthetic group in this enzyme
to form FADH2. This reaction also regenerates dihydroxyacetone
phosphate.
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Figure 18.37. Glycerol 3-Phosphate Shuttle. Electrons from NADH can enter
the mitochondrial electron transport chain by being used to reduce
dihydroxyacetone phosphate to glycerol 3-phosphate. Glycerol 3-phosphate is
reoxidized by electron transfer to an FAD prosthetic group in a membrane-bound
glycerol 3-phosphate dehydrogenase. Subsequent electron transfer to Q to form
QH2 allows these electrons to enter the electron-transport chain.
MalateAspartate
Shuttle.
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In the heart and liver, electrons from cytosolic NADH are brought into mitochondria by the
malate-aspartate shuttle, which is mediated by two membrane carriers and four enzymes.
Electrons are transferred from NADH in the cytosol to oxaloacetate, forming malate, which
traverses the inner mitochondrial membrane and is then reoxidized by NAD+ in the matrix to
form NADH in a reaction catalyzed by the citric acid cycle enzyme malate dehydrogenase. The
resulting oxaloacetate does not readily cross the inner mitochondrial membrane, and so a
transamination reaction is needed to form aspartate, which can be transported to the cytosolic
side. Mitochondrial glutamate donates an amino group, forming aspartate and α-ketoglutarate.
In the cytoplasm, aspartate is then deaminated to form oxaloacetate and the cycle is restarted.
This shuttle, in contrast with the glycerol 3-phosphate shuttle, is readily reversible.
Consequently, NADH can be brought into mitochondria by the malate- aspartate shuttle only if
the NADH/NAD+ ratio is higher in the cytosol than in the mitochondrial matrix. This versatile
shuttle also facilitates the exchange of key intermediates between mitochondria and the
cytosol.
The Entry of ADP into Mitochondria Is Coupled to the
Exit of ATP by ATP-ADP Translocase
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The major function of oxidative phosphorylation is
to generate ATP from ADP. However, ATP and ADP
do not diffuse freely across the inner mitochondrial
membrane. How are these highly charged
molecules moved across the inner membrane into
the cytosol? A specific transport protein, ATP-ADP
translocase (also called adenine nucleotide
translocase or ANT), enables these molecules to
traverse this permeability barrier. Most important,
the flows of ATP and ADP are coupled. ADP enters
the mitochondrial matrix only if ATP exits, and vice
versa. The reaction catalyzed by the translocase,
which acts as an antiporter.
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Mechanism of Mitochondrial ATP-ADP Translocase. The
translocase catalyzes the coupled entry of ADP and exit of ATP into
and from the matrix. The reaction cycle is driven by membrane
potential. The actual conformational change corresponding to
eversion of the binding site could be quite small.
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The membrane potential and hence the protonmotive force are decreased by the exchange of ATP
for ADP, which results in a net transfer of one
negative charge out of the matrix.
ATP/ADP exchange is energetically expensive;
about a quarter of the energy yield from electron
transfer by the respiratory chain is consumed to
regenerate the membrane potential that is trapped
by this exchange.
The inhibition of this process lead to the
subsequent inhibition of cellular respiration as well.
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Mitochondrial Transporters. Transporters (also called carriers)
are transmembrane proteins that move ions and charged metabolites
across the inner mitochondrial membrane.
Reaction sequence
ATP yield per
glucose molecule
Glycolysis: Conversion of glucose into pyruvate (in the cytosol)
Phosphorylation of glucose
-1
Phosphorylation of fructose 6-phosphate
-1
Dephosphorylation of 2 molecules of 1,3-BPG
+2
Dephosphorylation of 2 molecules of phosphoenolpyruvate
+2
2 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehyde 3-phosphate
Conversion of pyruvate into acetyl CoA (inside mitochondria)
2 molecules of NADH are formed
Citric acid cycle (inside mitochondria)
2 molecules of guanosine triphosphate are formed from 2 molecules of succinyl CoA
+2
6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, α-ketoglutarate, and malate
2 molecules of FADH2 are formed in the oxidation of 2 molecules of succinate
Oxidative phosphorylation (inside mitochondria)
2 molecules of NADH formed in glycolysis; each yields 1.5 molecules of ATP (assuming transport of NADH by the
glycerol 3-phosphate shuttle)
+3
2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP
+5
2 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules of ATP
+3
6 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP
+ 15
net yield per molecule of glucose
+ 30
The Pentose Phosphate
Pathway.
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Many endergonic reactions, notably the
reductive biosynthesis of fatty acids , and
cholesterol, as well as photosynthesis,
require NADPH in addition to ATP.
Despite their close chemical resemblance,
NADPH and NADH are not metabolically
interchangeable.
Whereas NADH participates in utilizing the
free energy of metabolite oxidation to
synthesize ATP (oxidative phosphorylation),
NADPH is involved in utilizing the free
energy of metabolite oxidation for
otherwise endergonic reductive
biosynthesis.
Pathways Requiring NADPH
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Synthesis:
–
–
–
–
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Fatty acid biosynthesis.
Cholesterol biosynthesis.
Neurotransmitter biosynthesis.
Nucleotide biosynthesis.
Detoxification:
– Reduction of oxidized glutathione.
– Cytochrome P50 monooxygenases.
NADPH is generated by the oxidation
of G6P via an alternative pathway to
glycolysis, the pentose phosphate
pathway (also called the hexose
monophosphate (HMP) shunt or the
phosphogluconate pathway.