Download The Citric Acid Cycle

Chapter 14
Citric Acid Cycle and Glyoxylate Cycle
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Biochemistry, 4th Edition
Chapter 14 Outline:
• Overview of Pyruvate Oxidation and the Citric Acid Cycle
• Pyruvate Oxidation: A Major Entry Route for Carbon into
the Citric Acid Cycle
• Coenzymes Involved in Pyruvate Oxidation and the Citric
Acid Cycle
• Action of the Pyruvate Dehydrogenase Complex
• The Citric Acid Cycle
• Stoichiometry and Energetics of the Citric Acid Cycle
• Regulation of Pyruvate Dehydrogenase and the Citric Acid
Cycle
• Anaplerotic Sequences: The Need to Replace Cycle
Intermediates
• Glyoxylate Cycle: An Anabolic Variant of the Citric Acid
Cycle
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Biochemistry, 4th Edition
Overview of Pyruvate Oxidation
and the Citric Acid Cycle
•
The citric acid cycle is the central
oxidative pathway in respiration, the
process by which all metabolic fuels—
carbohydrate, lipid, and protein—are
catabolized in aerobic organisms and
tissues.
•
Most of the energy yield from substrate
oxidation in the citric acid cycle comes
from subsequent reoxidation of reduced
electron carriers.
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Biochemistry, 4th Edition
Overview of Pyruvate Oxidation
and the Citric Acid Cycle
The three stages of respiration:
Stage 1 - carbon from metabolic fuels is
incorporated into acetyl-CoA.
Stage 2 - the citric acid cycle oxidizes
acetyl-CoA to produce CO2,
reduced electron carriers, and a
small amount of ATP.
Stage 3 - the reduced electron carriers are
reoxidized, providing energy for
the synthesis of additional ATP.
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Biochemistry, 4th Edition
Overview of Pyruvate Oxidation
and the Citric Acid Cycle
(a) Schematic of a mitochondrion.
(b) Computer model generated from
electron tomograms of a mitochondrion.
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•
Dehydrogenases catalyze substrate
oxidations.
•
Oxidases catalyze the subset of
oxidations in which O2 is the direct
electron acceptor.
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Biochemistry, 4th Edition
Overview of Pyruvate Oxidation
and the Citric Acid Cycle
The fate of carbon in the citric
acid cycle:
•Note that these departing CO2
groups derive from the two
oxaloacetate carboxyl groups that
were incorporated as acetyl-CoA in
earlier turns of the cycle.
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Biochemistry, 4th Edition
Pyruvate Oxidation: A Major Entry Route for
Carbon into the Citric Acid Cycle
•
Pyruvate oxidation to acetyl-CoA is catalyzed by the pyruvate
dehydrogenase complex (PDH complex).
•
It is an oxidative decarboxylation, which is virtually
irreversible involving three enzymes and five coenzymes.
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Biochemistry, 4th Edition
Pyruvate Oxidation: A Major Entry Route for
Carbon into the Citric Acid Cycle
Structure of the pyruvate dehydrogenase complex:
a)Electron micrograph of the purified pyruvate dehydrogenasecomplex from E.
coli.
b)E2 core subcomplex (60 E2 monomers).
c)E2-E3 subcomplex (E2 core 12 E3 dimers).
d)Full PDH complex (E2-E3 subcomplex 1 ~30 E1 tetramers).
e)Cutaway reconstruction of PDH complex.
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Biochemistry, 4th Edition
Pyruvate Oxidation: A Major Entry Route for
Carbon into the Citric Acid Cycle
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
•
In pyruvate oxidation, the acceptor of the active aldehyde
(hydroxyethyl group) generated by TPP is lipoic acid, which is the
internal disulfide of 6,8-dithiooctanoic acid.
•
The coenzyme is joined to via an amide bond linking the carboxyl
group of lipoic acid to a lysine e-amino group. Thus, the reactive
species is an amide, called lipoamide, or lipoyllysine.
•
Each lipoyllysine side chain is ~14Å long, and is located within a
flexible lipoyl domain of E2 (and E3BP), allowing it to function as a
“swinging arm” that can interact with the active sites of both the E1
and E3 components of the PDH complex.
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
Oxidized and reduced forms of lipoamide:
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•
The cyclic disulfide of lipoamide can undergo a
reversible two-electron reduction to form the
dithiol, dihydrolipoamide.
•
In pyruvate dehydrogenase, this reduction is
coupled to the transfer of the hydroxyethyl group
moiety from TPP, giving an acetyl thioester of the
reduced dihydrolipoamide.
•
Thus, lipoamide is a carrier of both electrons
and acyl groups.
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
•
Flavin coenzymes participate in two-electron oxidoreduction reactions that
can proceed in 2 one-electron steps.
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
•
Coenzyme A (A for acyl) participates in activation of acyl
groups in general, including the acetyl group derived from
pyruvate.
•
The coenzyme is derived metabolically from ATP, the vitamin
pantothenic acid, and b-mercaptoethylamine.
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
•
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Thioesters such as acetyl-CoA are energy rich because
thioesters are destabilized relative to ordinary oxygen
esters.
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Biochemistry, 4th Edition
Coenzymes Involved in Pyruvate Oxidation
and the Citric Acid Cycle
Comparison of free energies of hydrolysis of thioesters
and oxygen esters:
•
Lack of resonance stabilization in thioesters is the basis for the higher DG
of hydrolysis of thioesters, relative to that of ordinary oxygen esters.
•
The free energies of the hydrolysis products are similar for the two classes
of compounds.
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Biochemistry, 4th Edition
Action of the Pyruvate Dehydrogenase Complex
Mechanisms of the pyruvate dehydrogenase complex:
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Biochemistry, 4th Edition
Action of the Pyruvate Dehydrogenase Complex
•
Lipoamide is tethered to one enzyme (E2) in the PDH complex, but
it interacts with all three enzymes via a flexible swinging arm.
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Biochemistry, 4th Edition
Action of the Pyruvate Dehydrogenase Complex
•
Arsenic poisoning, both intentional and unintentional, has had a
long history, dating back to at least the eighth century.
•
Trivalent As(III) compounds such as arsenite and organic
arsenicals react readily with thiols, and they are especially
reactive with dithiols, such as dihydrolipoamide, forming
bidentate adducts:
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 1: Introduction of Two Carbon Atoms as Acetyl-CoA
•
The initial reaction, catalyzed by citrate synthase, is akin to an
aldol condensation.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Mechanism of the citrate synthase reaction:
Step 1: Asp 375 extracts a proton from the methyl group,
and His 274 donates a proton to the carbonyl oxygen
of acetyl-CoA, creating an enol.
Step 2: His 274 deprotonates the acetyl-CoA enol, stabilizing
the nucleophilic enolate that attacks the keto carbon of
oxaloacetate. His 320 protonates the aldol product (S)citroyl-CoA.
Step 3: The citroyl-CoA intermediate spontaneously hydrolyzes
to citrate by a nucleophilic acyl substitution reaction
giving exclusively the S stereoisomer of citroyl-CoA.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Three-dimensional structure of citrate synthase:
•The two forms of pig heart citrate synthase homodimer shown here were
determined by crystallographic methods and support the induced fit model of
enzyme catalysis.
(a)In the absence of CoA-SH the enzyme crystallizes in an “open” form. Citrate
binds at the base of large clefts in both catalytic domains of the homodimeric
protein.
(b)Binding of CoA-SH causes the enzyme to adopt a “closed” conformation, with
the clefts essentially filled.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 2: Isomerization of Citrate
•The tertiary alcohol of citrate presents yet another chemical
problem: tertiary alcohols cannot be oxidized without breaking a
carbon–carbon bond.
•To set up the next oxidation in the pathway, citrate is converted
to isocitrate, a chiral secondary alcohol, which can be more
readily oxidized.
•This isomerization reaction, catalyzed by aconitase, involves
successive dehydration and hydration, through cis-aconitate as
a dehydrated intermediate, which remains enzyme-bound.
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Biochemistry, 4th Edition
The Citric Acid Cycle
•
The enzyme contains nonheme iron and acid-labile sulfur in a
cluster called a 4Fe–4S iron–sulfur center.
•
The iron–sulfur cluster coordinates the hydroxyl group and one of
the carboxyl groups on the citrate molecule.
•
The cis-aconitate intermediate must flip 180° during the reaction,
presumably by releasing from the enzyme, then rebinding with the
iron–sulfur cluster, but in the opposite orientation.
•
Thus, of the four possible diastereomers of isocitrate, only one, the
2R,3S diastereomer, is produced.
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Biochemistry, 4th Edition
The Citric Acid Cycle
The prochirality of citrate when bound to aconitase:
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Biochemistry, 4th Edition
The Citric Acid Cycle
•
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Fluoroacetate is an example of a mechanism-based, or
suicide, inhibitor of aconitase.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 3: Generation of CO2 by an NAD+-Linked Dehydrogenase
•The first of two oxidative decarboxylations in the cycle is catalyzed by
isocitrate dehydrogenase.
•Isocitrate is oxidized to a ketone, oxalosuccinate, an unstable
•enzyme-bound intermediate that spontaneously b-decarboxylates to give the
product, a-ketoglutarate.
•The strategy here is to oxidize isocitrate’s secondary alcohol to a keto group
b to the carboxyl group to be removed.
•The b-keto group acts as an electron sink to stabilize the carbanionic
transition state, facilitating decarboxylation.
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Biochemistry, 4th Edition
The Citric Acid Cycle
•
Two carbon atoms enter the citric acid cycle as acetyl-CoA, and
two are lost as CO2 in the oxidative decarboxylations of steps 3
and 4.
Step 4: Generation of a Second CO2 by an Oxidative
Decarboxylation
•
This is a multistep reaction entirely analogous to the pyruvate
dehydrogenase reaction.
•
An a-keto acid substrate undergoes oxidative decarboxylation,
with concomitant formation of an acyl-CoA thioester.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Decarboxylation of a-ketoglutarate:
•The first step catalyzed out by the a-ketoglutarate
dehydrogenase complex is a decarboxylation catalyzed by aketoglutarate decarboxylase (E1 of the complex), producing a
four-carbon TPP derivative.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 5: A Substrate-Level Phosphorylation
•
Succinyl-CoA is an energy-rich thioester compound, and its
potential energy is used to drive the formation of a nucleoside
triphosphate
•
This reaction, catalyzed by succinyl-CoA synthetase, is
comparable to the two substrate-level phosphorylation reactions
that we encountered in glycolysis, except that in animal cells the
energy-rich nucleotide product is not always ATP but, in some
tissues, GTP.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Covalent catalysis by the succinyl-CoA
synthetase reaction:
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•
Three successive nucleophilic
substitution reactions conserve the
energy of the thioester of succinyl-CoA
in the phosphoanhydride bond of ATP
(or GTP).
•
An active site histidine side chain is
transiently phosphorylated (Nphosphohistidine) during the reaction.
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Biochemistry, 4th Edition
The Citric Acid Cycle
•
A charge–dipole interaction
stabilizes the phosphohistidine
intermediate in the succinyl-CoA
synthetase reaction.
•
In this schematic, based on the E.
coli enzyme structure, the
permanent dipoles of two -helices
(the “power” helices) are oriented
such that the d+ at their N-termini
interact with the negative charges
on the phosphate group of the
active site N-phosphohistidine,
stabilizing this transient reaction
intermediate.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 6: A Flavin-Dependent Dehydrogenation
•
Completion of the cycle involves conversion of the four-carbon
succinate to the four-carbon oxaloacetate.
•
The first of the three reactions, catalyzed by succinate
dehydrogenase, is the FAD-dependent dehydrogenation of two
saturated carbons to a double bond.
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Biochemistry, 4th Edition
The Citric Acid Cycle
•
Succinate dehydrogenase is competitively inhibited by
malonate, a structural analog of succinate.Malonate inhibition
of pyruvate oxidation was one of the clues that led Krebs to
propose the cyclic nature of this pathway.
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Biochemistry, 4th Edition
The Citric Acid Cycle
•
A single C-C bond is more difficult to oxidize than a C-O bond.
•
Therefore, the redox coenzyme for succinate dehydrogenase
is not but the more powerful oxidant FAD.
•
The flavin is bound covalently to the enzyme protein through a
specific histidine residue.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 7: Hydration of a Carbon–Carbon Double Bond
•The stereospecific trans hydration of the carbon–carbon double
bond is catalyzed by fumarate hydratase, more commonly
called fumarase.
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Biochemistry, 4th Edition
The Citric Acid Cycle
Step 8: A Dehydrogenation that Regenerates
Oxaloacetate
•Finally, the cycle is completed with the NAD+–dependent
dehydrogenation of malate to oxaloacetate, catalyzed by malate
dehydrogenase.
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Biochemistry, 4th Edition
Stoichiometry and Energetics
of the Citric Acid Cycle
•
One turn of the citric acid cycle generates one high-energy phosphate (ATP or GTP)
through substrate-level phosphorylation, plus three NADH and one FADH2 for
subsequent reoxidation in the electron transport chain.
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Biochemistry, 4th Edition
Regulation of Pyruvate Dehydrogenase
and the Citric Acid Cycle
Major regulatory factors controlling
pyruvate dehydrogenase
and the citric acid cycle:
•
Red brackets indicate concentration
dependence.
•
NADH can inhibit through allosteric
interactions, but apparent NADH
inhibition can also be a reflection of
reduced NAD+ availability.
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Biochemistry, 4th Edition
Regulation of Pyruvate Dehydrogenase
and the Citric Acid Cycle
•
Regulation of the mammalian
pyruvate dehydrogenase complex
by feedback inhibition and by
covalent modification of E1.
•
A kinase and a phosphatase
inactivate and activate the first
component (E1) of the pyruvate
dehydrogenase complex by
phosphorylating and
dephosphorylating, respectively,
three specific serine residues.
•
The active form of the pyruvate
dehydrogenase complex is
feedback inhibited by acetyl-CoA
and NADH.
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Biochemistry, 4th Edition
Regulation of Pyruvate Dehydrogenase
and the Citric Acid Cycle
•
The citric acid cycle is controlled primarily by the relative
intramitochondrial concentrations of NAD+ and NADH.
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Biochemistry, 4th Edition
Anaplerotic Sequences:
The Need to Replace Cycle Intermediates
•
Citric acid cycle intermediates used in biosynthetic
pathways must be replenished to maintain flux through the
cycle.
•
Anaplerotic pathways serve this purpose.
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Biochemistry, 4th Edition
Anaplerotic Sequences:
The Need to Replace Cycle Intermediates
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Biochemistry, 4th Edition
Anaplerotic Sequences:
The Need to Replace Cycle Intermediates
•
Because phosphoenolpyruvate is such an energy-rich
compound, this reaction, catalyzed by
phosphoenolpyruvate carboxylase, requires neither an
energy cofactor nor biotin. This reaction is important in the C4
pathway of photosynthetic CO2 fixation
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Biochemistry, 4th Edition
Anaplerotic Sequences:
The Need to Replace Cycle Intermediates
•
In addition to pyruvate carboxylase and phosphoenolpyruvate
carboxylase, a third anaplerotic process is provided by an
enzyme commonly known as malic enzyme but more officially as
malate dehydrogenase (decarboxylating:NADP+).
•
The malic enzyme catalyzes the reductive carboxylation of
pyruvate to give malate.
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Biochemistry, 4th Edition
Anaplerotic Sequences:
The Need to Replace Cycle Intermediates
Mechanism of the biotin-dependent pyruvate carboxylase reaction:
•
•
Phase I is catalyzed by the biotin carboxylation (BC) domain.
Phase II is catalyzed by the carboxyltransferase (CT) domain.
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Biochemistry, 4th Edition
Anaplerotic Sequences:
The Need to Replace Cycle Intermediates
Mechanism of the biotin-dependent pyruvate carboxylase reaction:
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Biochemistry, 4th Edition
Glyoxylate Cycle: An Anabolic Variant
of the Citric Acid Cycle
•
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The glyoxylate cycle allows plants and
bacteria to carry out net conversion of
fat to carbohydrate, bypassing CO2generating reactions of the citric acid
cycle.
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Biochemistry, 4th Edition
Glyoxylate Cycle: An Anabolic Variant
of the Citric Acid Cycle
Reactions of the glyoxylate cycle:
•
•
•
Two acetyl-CoA molecules enter the
cycle, one at the citrate synthase
step, and the second at the malate
synthase step.
The reactions catalyzed by isocitrate
lyase and malate synthase bypass
the three citric acid cycle steps
between isocitrate and succinate so
that the two carbons lost in the citric
acid cycle are saved, resulting in the
net synthesis of oxaloacetate.
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Biochemistry, 4th Edition
Glyoxylate Cycle: An Anabolic Variant
of the Citric Acid Cycle
Intracellular relationships involving
the glyoxylate cycle in plant cells:
•
Fatty acids released in lipid bodies
are oxidized in glyoxysomes to
acetyl-CoA, which can also come
directly from acetate.
•
Acetyl-CoA is then converted to
succinate in the glyoxylate cycle,
and the succinate is transported to
mitochondria.
•
There it is converted in the citric
acid cycle to oxaloacetate, which
is readily converted to sugars by
gluconeogenesis.
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