1. Chapter 16 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 oxidation and to participate in citric acid synthesis.
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4. ? Acetyl-CoA was discovered only in 1951 The original
acetyl phosphate
acetyl phosphate
The original
citric acid cycle
Krebs and Johnson (1937) “The role of citric acid in the intermediate
metabolism in animal tissues. Enzymologica 4:
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This paper was submitted to Nature but was rejected!!!

5. 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!
For Many years,
Lipmann believed
that Acetyl-phosphate,
instead of acetyl-CoA,
is the two-carbon “active” compound condensing
with oxaloacetate.
Hans Adolf Krebs
( )
Fritz Albert Lipmann
( )
(both were born
In Germany)
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United Kingdom
USA

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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 H+ gradient, which will promote ATP formation.
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7. Mitochondria was found to be the major site for fuel oxidation to
generate ATP.
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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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9. The oxidative decarboxylation of pyruvate in mitochondria.
Reaction irreversible
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 degradation or biosynthesis.
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11. Lipoate acts as both electron and acyl carriers.
硫辛酸
Lipoate acts as both electron
and acyl carriers.
It swings between the three
different active sites of the
pyruvate dehydrogenase complex.
“The arm”
(~14 A)
Arsenic compounds are poisonous
because they sequester lipoamide.
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12. The five reactions catalyzed by pyruvate dehydrogenase complex:
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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: E1 E2
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15. The citric acid cycle consists of eight successive reactions
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16. 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)
(柠檬酸)
Hydrolysis of the thioester bond
releases a large amount of free energy.
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Citrate synthase before binding to oxaloacetate
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Citrate synthase after binding to oxaloacetate
Acetyl-CoA analog
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Active site is located at the interface of the two subunits.

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A prochiral molecule
顺乌头酸酶
异柠檬酸
Aconitase then catalyzes
the interconversion
of citrate and isocitrate
via dehydration and
hydration.
Aconitase contains
an iron-sulfur center
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Isocitrate is then converted to a-ketoglutarate via oxidative decarboxylation, producing CO2.
a-酮戊二酸
Oxalosuccinate is an intermediate; the carbon released as CO2 did
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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23. The only substrate-level phosphorylation in the cycle
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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24. 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
citric acid cycle.
(An alkene)
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25. Malonate is a strong competitive inhibitor of succinate dehydrogenase
丙二酸
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26. 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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27. Neither maleate nor D-malate are substrates of fumarase
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28. 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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30. “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
between acetate and oxaloacetate!
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The symmetric molecule can react asymmetrically in the asymmetric active site of enzymes. X
Active site of aconitase
Citrate is a prochiral molecule
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32. 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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36. The citric acid cycle intermediates need to be replenished
Activated by acetyl-CoA
Activated by Fru-1,6P
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37. 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!
Some modern anaerobic microorganisms use an incomplete citric acid cycle as a source of biosynthetic precursors, not of energy!
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40. Most of the regulation is provided by substrate
Activity of pyruvate
dehydrogenase
complex is regulated
by allosteric and
covalent mechanisms.
Phosphorylated
E1 is inactive.
Most of the regulation is
provided by substrate
availability and product
inhibition.
Inhibits PFK-1
Three enzymes of the citric
acid cycle are regulated to
maintain a steady state level
of ATP for the cells.
Enzymes are switched off
when the energy charge is
high and biosynthetic
intermediates are abundant.
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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 hexoses.
H.L. Kornberg and H.A. Krebs, Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature 179 (1957), pp. 988–991
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42. The two decarboxylation steps of the citric acid
The glyoxylate
cycle
苹果酸合酶
异柠檬酸裂解酶
The two decarboxylation
steps of the citric acid
cycle are bypassed.
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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.
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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 eight-step 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 biotin-containing pyruvate carboxylase.
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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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