Download The Citric Acid Cycle

For Biochemistry II lecture of September 25, 2007
Chapter 16
The Citric Acid Cycle
A central metabolic hub, with catabolic
pathways leading in and anabolic
pathways leading out.
More than 95% of the energy for the human being
is generated through this pathway (in conjunction
with the oxidative phosphorylation process).
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Carbon compounds that might be
intermediates in the intracellular
oxidation of pyruvate to CO2 and
H2O were searched in the 1930s

Four-carbon dicarboxylic acids (first fumarate, then
succinate, malate, oxaloacetate) were found to catalyze
the O2 uptake by suspensions of minced pigeon-breast
muscle (Szent-Gyogyi, 1935); Then six-carbon
tricarboxylic citric acid was also found to exert a similar
catalytic effect (Krebs and Johnson, 1937).
 Malonate was found to inhibit pyruvate oxidation
regardless of which active organic acid is added!
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
Carbon compounds that might
be intermediates in the
intracellular oxidation of
pyruvate to CO2 and H2O were
searched in the 1930s
Citrate was found to be synthesized from oxaloacetate
and a substance which could be derived from
carbohydrate, like pyruvate or acetate (Krebs and
Johnson, 1937).
 A complete scheme of carbohydrate oxidation was thus
formulated (Krebs and Johnson, 1937).
 Coenzyme A (A for acetylation) was found as a
cofactor for the acetylation reactions (Lipmann, 1945).
 Coenzyme A was then found to be needed for pyruvate
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oxidation and to participate
in citric acid synthesis.
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?
Acetyl-CoA was
discovered only in 1951
acetyl phosphate
The original
citric acid cycle
Krebs and Johnson (1937) “The role of citric acid in the intermediate
metabolism in animalDownloaded
tissues. from
Enzymologica 4:148-156.
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This paper was submitted
to Nature but was rejected!!!
The Nobel Prize in Physiology or Medicine 1953
"for his discovery of co-enzyme A
and its importance for intermediary
metabolism"
"for his discovery
of the citric acid cycle"
Krebs discovered
the urea cycle in
1932 before he
elucidated
the citric acid
cycle!
Hans Adolf Krebs
(1900-1981)
United Kingdom
For Many years,
Lipmann believed
that Acetyl-phosphate,
instead of acetyl-CoA,
is the two-carbon
“active” compound
condensing
with oxaloacetate.
(both were
born
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Germany)
Fritz Albert Lipmann
(1899-1986)
USA
The cellular respiration (complete
oxidation of most fuels) can be
divided into three stages

Stage I All the fuel molecules are oxidized to
generate a common two-carbon unit, acetyl-CoA.
 Stage II The acetyl-CoA is completely oxidized
into CO2, with electrons collected by NAD+ and
FAD via a cyclic pathway (the citric acid cycle,
Krebs cycle, or tricarboxylic acid cycle).
 Stage III Electrons of NADH and FADH2 are
transferred to O2 via the respiratory chain (a
series of electron carriers), producing H2O and a
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+
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H gradient, which
will promote ATP formation.
Mitochondria was found to be the
major
site
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generate ATP.
Pyruvate is first transported into
mitochondria via a specific
transporter on the inner
membrane and then oxidized to
acetyl-CoA by the catalysis of
pyruvate dehydrogenase
complex.
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The oxidative decarboxylation of
irreversible
pyruvateReaction
in mitochondria.
Stoichiometric
cofactors
Catalytic
cofactors
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Coenzyme A was first discovered
by Lipmann in 1945.
辅酶A
Coenzyme A (CoA-SH) delivers activated
acyl groups (with 2-24 carbons) for
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degradation
or biosynthesis.
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硫辛酸
“The arm”
(~14 A)
Lipoate acts as both electron
and acyl carriers.
It swings between the three
different active sites of the
pyruvate dehydrogenase complex.
Arsenic compounds are poisonous
because they sequester lipoamide.
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The five reactions catalyzed by
pyruvate dehydrogenase complex:
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E1
E2
An icosahedral structure, based on
electron cryo-microscopic analysis
of an E1E2 sub-complex from
B.stearothermophilus. Three of the
60 E2 molecules (colored red, green
and yellow) are highlighted. The
movement of the swinging E2
lipoyl domain in the annular
region between the inner core
(cyan) of E2 molecules and the
outer shell of E1 molecules
(purple) is proposed to be a critical
feature underlying active site
coupling in the complex.
Milne et al., 2002, EMBO J.
21:5587-5598
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The citric acid cycle
consists of eight
successive
reactions
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The cycle begins with the
condensation of acetyl-CoA and
oxaloacetate to form citrate
Aldol condensation
followed by a hydrolysis
柠檬酸合酶
(草酰乙酸)
Hydrolysis of the thioester
bond
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releases a large amount
of free energy.
(柠檬酸)
Citrate synthase before binding to oxaloacetate
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Citrate synthase after binding to oxaloacetate
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Acetyl-CoA analog
Active site is located at the interface of the two subunits.
A prochiral molecule
顺乌头酸酶
异柠檬酸
Aconitase contains
an iron-sulfur center
Aconitase then catalyzes
the interconversion
of citrate and isocitrate
via dehydration and
hydration.
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Isocitrate is then converted to aketoglutarate via oxidative
decarboxylation, producing CO2.
a-酮戊二酸
Oxalosuccinate is an intermediate; the carbon released as CO2 did
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not come from the acetyl-CoA
entering
the cycle.
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The a-ketoglutarate is then converted to
succinyl-CoA via another oxidative
decarboxylation, producing the second
CO2.
TPP
lipoate
FAD
琥珀酰辅酶A
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Succinyl-CoA is then converted
to succinate, accompanied by
the formation of a GTP (or ATP)
Synthetase(合成酶): ATP-consuming;
Synthase(合酶): not ATP-consuming.
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Proposed mechanism
for the succinyl-CoA
synthetase catalyzed
GDP phosphorylation,
via a phosphoenzyme
intermediate.
A phosphohistidine
intermediate in the reaction
convinced many biochemists in
the 1950's that all of oxidative
phosphorylation was due to
chemical coupling instead of
chemiosmotic coupling!
An acyl phosphate
ATP
The only substrate-level
phosphorylation in the cycle
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Succinate is then converted to
fumarate via dehydrogenation
延胡索酸
(An alkane)
A flavoprotein with a covalently bound
FAD and three iron-sulfur centers; the
only integral membrane protein
for the
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citric acid cycle.
(An alkene)
Malonate is a strong competitive
inhibitor of succinate dehydrogenase
丙二酸
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Fumarate is then converted to
L-malate via hydration
A stereospecific enzyme only
acting on the trans and L isomers
苹果酸
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Neither maleate nor D-malate
are substrates of fumarase
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The cycle ends by the regeneration of
oxaloacetate from L-malate
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None of the intermediates
are phosphorylated.
All are either di- or
tricarboxylic acids.
An overview of
the citric acid cycle
3
To regenerate
oxaloacetate.
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“unexpected” results of an early
isotope labeling experiment
Chemically indistinguishable
Citrate: A symmetrical molecule
That reacts asymmetrically
Cis-aconitate or isocitrate, instead of citrate,
was proposed to be the first condensation product
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between acetate and oxaloacetate!
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The symmetric molecule
can react asymmetrically
in the asymmetric active
site of enzymes.
X
Citrate is a prochiral molecule
Active site of aconitase
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Overall efficiency
of energy conservation:
65%
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Accumulating evidence also
suggest the formation of
multienzyme complexes (or
metabolons) for the citric
acid cycle enzymes,
facilitating substrate
channeling between the
active sites.
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The citric acid
intermediates are
important sources for
biosynthetic precursors.
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The citric acid cycle
is amphibolic.
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The citric acid cycle intermediates
need to be replenished
Activated by acetyl-CoA
Activated by Fru-1,6P
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A proposed mechanism for pyruvate carboxylase:
biotin acts as a tethered long arm that carries the
activated CO2.
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Common biological tethers found in proteins
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a-ketoglutarate
dyhydrogenase
is not present in
such organisms!
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Some modern
anaerobic
microorganisms
use an
incomplete citric
acid cycle as a
source of
biosynthetic
precursors, not
of energy!
Activity of pyruvate
dehydrogenase
complex is regulated
by allosteric and
covalent mechanisms.
Phosphorylated
E1 is inactive.
Enzymes are switched off
when the energy charge is
high and biosynthetic
intermediates are abundant.
Inhibits PFK-1
Most of the regulation is
provided by substrate
availability and product
inhibition.
Three enzymes of the citric
acid cycle are regulated to
maintain a steady state level
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of ATP for the cells.
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Net conversion of acetate to carbohydrates is
allowed via the glyoxylate cycle occurring in
certain organisms

There is no net conversion of acetate (also from fatty acids
and amino acids) to any of the citric acid cycle intermediate,
thus neither to carbohydrates.
 Net conversion of acetate to four-carbon citric acid cycle
intermediates occurs via the glyoxylate cycle, occurring in
plants, certain invertebrates, and some microorganisms
(including E. coli and yeast).
 The glyoxylate cycle was first discovered in bacteria by
Kornberg & Krebs in 1957 as a means of converting C2
units of acetate (a growth substrate) for synthesis of other
cell constituents such as Downloaded
hexoses.
from
H.L. Kornberg and H.A. Krebs, Synthesis of cell constituents from C2-units by a modified tricarboxylic
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acid cycle. Nature 179 (1957), pp. 988–991
The glyoxylate
cycle
苹果酸合酶
异柠檬酸裂解酶
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The two decarboxylation
steps of the citric acid
cycle are bypassed.
Partial overlapping
of the glyoxylate and
citric acid cycles
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Conversion of fatty acids to glucose (in
germinating seeds) occurs in three
intracellular compartments
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Partition of
isocitrate between
the glyoxylate and
citric acid cycles
are regulated by
the opposite effect
of common
allosteric effectors
on the isocitrate
lyase and the
isocitrate
dehydrogenase.
Summary

Pyruvate is converted to acetyl-CoA by the
action of pyruvate dehydrogenase complex, a
huge enzyme complex.
 Acetyl-CoA is converted to 2 CO2 via the eightstep citric acid cycle, generating three NADH,
one FADH2, and one ATP (by substrate-level
phophorylation).
 Intermediates of citric acid cycle are also used
as biosynthetic precursors for many other
biomolecules, including fatty acids, steroids,
amino acids, heme, pyrimidines, and glucose.
 Oxaloacetate can get replenished from pyruvate,
via a carboxylation reaction
catalyzed by the
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pyruvate carboxylase.

The activity of pyruvate dehydrogenase complex
is regulated by allosteric effectors and reversible
phosphorylations.
 Net conversion of fatty acids to glucose can occur
in germinating seeds, some invertebrates and
some bacteria via the glycoxylate cycle, which
shares three steps with the citric acid cycle but
bypasses the two decarboxylation steps,
converting two molecules of acetyl-CoA to one
succinate.
 Acetyl-CoA (isocitrate) is partitioned into the
glyoxylate cycle and citric acid cycle via a
coordinately regulation of the isocitrate
dehydrogenase and isocitrate
lyase.
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