Transcript The Citric Acid Cycle

Chapter 16
The Citric Acid Cycle
The common pathway leading to complete
oxidation of carbohydrates, fatty acids, and
amino acids to CO2.
A pathway providing many precursors for
biosynthesis
1. The cellular respiration
(complete oxidation of 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 (named as the citric acid cycle,
Krebs cycle, or tricarboxylic acid cycle).
• Stage III Electrons of NADH and FADH2 are
transferred to O2 via a series carriers, producing H2O
and a H+ gradient, which will promote ATP
formation.
Mitochondria is the major site for
fuel oxidation to generate ATP.
2. Pyruvate is oxidized to acetylCoA by the catalysis of pyruvate
dehydrogenase complex
• Pyruvate is first transported into mitochondria via a
specific transporter on the inner membrane.
• Pyruvate is converted to acetyl-CoA and CO2 by
oxidative decarboxylation.
• The pyruvate dehydrogenase complex is a huge
multimeric assembly of three kinds of enzymes,
having 60 subunits in bacteria and more in mammals.
• Pyruvate is first decarboxylated after binding to the
prosthetic group (TPP) of pyruvate dehydrogenase
(E1), forming hydroxyethyl-TPP.
• The hydroxyethyl group attached to TPP is oxidized
and transferred: First two electrons, then the acetyl
group formed are all transferred to the lipoyllysyl
(硫辛酰赖氨酰）group of dihydrolipoyl
transacetylase (E2).
• The lipoyllysyl group serves as both electron and
acetyl carriers.
• The acetyl group is then transferred (still catalyzed
by E2) from acetyllipoamide to CoA-SH,forming
acetyl-CoA.
• The oxidized lipoamide group is then regenerated by
the action of dihydrolipoyl dehydrogenase (E3), with
electrons collected by FAD and then by NAD+.
• Substrates of the five reactions catalyzed by
pyruvate dehydrogenase complex are efficiently
channeled : The lipoamide group attached to E2
swings between E1 (accepting the electrons and
acetyl group) and E3 (giving away the electrons),
passing the acetyl group to Coenzyme A on E2
• The multienzyme complexes catalyzing the
oxidative decarboxylation of a few different kinds of
a-keto acids, pyruvate dehydrogenase complex, aketoglutarate dehydrogenase complex and branched
chain a-keto acid dehydrogenase complex show
remarkable structure and function relatedness (all
have identical E3, similar E1 and E2).
The oxidative decarboxylation of pyruvate
in mitochondria: producing acetyl-CoA and CO2.
Electron micrograph of pyruvate
dehydrogenase complexes from E. coli
pyruvate
CO2 acetyl-CoA
hydroxyethyl-TPP
E2
E3
E2 (dihydrolipoyl transacetylase):
consisting the core, 24 subunits;
E1 (pyruvate dehydrogenase):
bound to the E2 core, 24 subunits;
E3 (dihydrolipoyl dehydrogenase):
bound to the E2 core, 12 subunits.
(a protein kinase and
phosphoprotein phosphatase, not
shown here, are also part of the
complex)
A model of the E. coli pyruvate dehydrognase
complex showing the three kinds of enzymes and
the flexible lipoamide arms covalently attached to E2
The E2 core
(a total of 24 subunits)
forms a hollow
cube.
X-ray structure of the E2 transacetylase core: Only
four out of eight trimers are shown here.
The oxidative decarboxylation of pyruvate is catalyzed by
a multiezyme complex: pyruvate dehydrogenase complex.
With the help of TPP, pyruvate is decarboxylated:
identical reaction as catalyzed by pyruvate decarboxylase.
Dihydrolipoyl
The lipoyllysyl group
serves as the lectron
and acetyl carriers
Coenzyme A (CoA-SH): discovered in 1945 by
Lipmann, one of the “carrier molecules”,
deliver activated acyl groups (with 2-24
Carbons) for degradation or biosynthesis.
3. The complete oxidation of pyruvate
in animal tissues was proposed to
undergo via a cyclic pathway
• O2 consumption and pyruvate oxidation in minced
muscle tissues were found to be stimulated by some
four-carbon dicarboxylic acids (Fumarate, succinate,
malate and oxaloacetate, five-carbon dicarboxylic
acid (a-ketoglutarate ), or six-carbon tricarboxylic
acids (citrate, isocitrate, cis-aconitate).
• A small amount of any of these organic acids
stimulates many folds of pyruvate oxidation!
• Malonate inhibits pyruvate oxidation regardless of
which active organic acid is added!
• Hans Krebs proposed the “citric acid cycle” for the
complete oxidation of pyruvate in animal tissues in
1937 (he wrongly hypothesized that pyruvate
condenses with oxaloacetate in his original
proposal).
• The citric acid cycle was confirmed to be universal
in cells by in vitro studies with purified enzymes and
in vivo studies with radio isotopes (“radio isotope
tracer experiments”).
• Krebs was awarded the Nobel prize in medicine in
1953 for revealing the citric acid cycle (thus also
called the Krebs cycle).
4. The acetyl group (carried by CoA)
is completely oxidized to CO2 via
the citric acid cycle
• The 4-carbon oxaloacetate (草酰乙酸) acts as the
“carrier” for the oxidation.
• The two carbons released as 2 CO2 in the first
cycle of oxidation are not from the acetyl-CoA just
joined.
• The 8 electrons released are collected by three
NAD+ and one FAD.
• One molecule of ATP (or GTP) is produced per
cycle by substrate-level phosphorylation.
The citric
acid cycle
5. The citric acid cycle consists
of eight successive reactions
• Step 1 The methyl carbon of acety-CoA joins the
carbonyl carbon of oxaloacetate via aldol
condensation to form citrate (柠檬酸); citroyl-CoA
is a transiently intermediate but hydrolyzed
immediately in the active site of citrate synthase;
hydrolysis of the thioester bond releases a large
amount of free energy, driving the reaction forward;
large conformational changes occur after
oxaloacetate is bound and after citroyl-CoA is
formed, preventing the undesirable hydrolysis of
acetyl-CoA.
• Step 2 Citrate is isomerized into isocitrate (get the
six-carbon unit ready for oxidative decarboxylation)
via a dehydration step followed by a hydration step;
cis-aconitate (顺乌头酸) is an intermediate during
this transformation, thus the catalytic enzyme is
named as aconitase, which contains a 4Fe-4S ironsulfur center directly participating substrate binding
and catalysis.
• Step 3 Isocitrate is first oxidized and then
decarboxylated to form a-ketoglutarate (a-酮戊二
酸); oxalosuccinate is an intermediate; two electrons
are collected by NAD+; the carbon released as CO2
is not from the acetyl group joined; catalyzed by
isocitrate dehydrogenase.
• Step 4 a-ketoglutarate undergoes another round of
oxidative decarboxylation; decarboxylated first, then
oxidized to form succinyl-CoA (琥珀酰辅酶A);
again the carbon released as CO2 is not from the
acetyl group joined; catalyzed by a-ketoglutarate
dehydrogenase complex; reactions and enzymes
closely resemble pyruvate dehydrogenase complex
(with similar E1 and E2, identical E3).
• Step 5 Succinyl-CoA is hydrolyzed to succinate (琥
珀酸或戊二酸); the free energy released by
hydrolyzing the thioester bond is harvested by a
GDP or an ADP to form a GTP or an ATP by
substrate-level phosphorylation; the reversible
reaction is catalyzed by succinyl-CoA synthetase (or
succinic thiokinase);
acyl phosphate and phophohistidyl enzyme are
intermediates; the active site is located at the
interface of two subunits; the negative charge of
the phospho-His intermediate is stabilized by the
electric dipoles of two a helices (one from each
subunit).
Step 6 Succinate is oxidized to fumarate (延胡索酸
或反丁烯二酸); catalyzed by a flavoprotein
succinate dehydrogenase (with a covalently bound
FAD and three iron-sulfur centers), which is
tightly bound to the inner membrane of
mitochondria; malonate (丙二酸) is a strong
competitive inhibitor of the enzyme, that will
block the whole cycle.
Step 7 Fumarate is hydrated to L-malate by the
action of fumarase; the enzyme is highly
stereospecific, only act on the trans and L isomers,
not on the cis and D isomers (maleate and Dmalate);
Step 8 Oxaloacetate is regenerated by the oxidation
of L-malate; this reaction is catalyzed by malate
dehydrogenase with two electrons collected by
NAD+.
The aldol condensation between acetyl-CoA and
oxaloacetate forms citrate
Citrate synthase before
and after binding to
oxaloacetate
Oxaloacetate
Carboxylmethyl-CoA
Citrate is converted to isocitrate via dehydration followed by a
hydration step.
4Fe-4S cubic array:
each Fe is bonded
to three inorganic
S and a cysteine
sulfur atom (except
one)
The first
oxidation step
Isocitrate is converted to a-ketoglutarate via an
oxidative decarboxylation step, generating NADH
CO2.
TPP
lipoate
FAD
(E1, E2, E3)
The second
oxidation step
The a-ketoglutarate dehydrogenase complex
closely resembles the pyruvate dehyrogenase
complex in structure and function
Succinyl-CoA synthetase
catalyzes the substrate-level
phosphorylation of ADP.
Succinyl-CoA
Synthetase from
E. coli
Coenzyme A
His246-Pi
The power helices
The third oxidation step
(An enzyme bound to
the inner membrane
of mitochondria)
(a stereospecific
enzyme)
(The fourth oxidation
Step in the cycle)
Oxaloacetate is regenerated at the end
5. The complete oxidation of one
glucose may yield as many as 32 ATP
• All the NADH and FADH2 will eventually pass their
electrons to O2 after being transferred through a
series of electron carriers.
• The complete oxidation of each NADH molecule
leads to the generation of about 2.5 ATP, and FADH2
of about 1.5 ATP.
• Overall efficiency of energy conservation is about
34% using the free energy changes under standard
conditions and about 65% using actual free energy
changes in cells.
6. The citric acid intermediates are
important sources for biosynthetic
precursors
• The citric acid cycle is the hub of intermediary
metabolism serving both the catabolic and anabolic
processes (thus an amphibolic pathway).
• It provides precursors for the biosynthesis of glucose,
amino acids, nucleotides, glucose, fatty acids,sterols,
heme groups, etc.
• Intermediates of the citric acid cycle get replenished
by anaplerotic reactions when consumed by
biosynthesis.
• The most common anaplerotic reactions covert
•
•
•
•
either pyruvate or phosphoenolpyruvate to
oxaloacetate or malate.
Pyruvate carboxylase catalyzes the carboxylation of
pyruvate using covalently bound biotin coenzyme.
The activated CO2 is first attached to an ureido N on
biotin and then transferred to the ionized enolate
form of pyruvate, producing oxaloacetate.
The long flexible arm of biotin switches between
two active site of the pyruvate carboxylase (one for
attaching a HCO3- to biotin and the other for
transferring the carboxyl group to pyruvate).
Acetyl-CoA is a positive allosteric modulator for the
carboxylase.
• Some anaerobic bacteria, lacking the a-ketoglutarate
dehydrogenase enzyme, make biosynthetic
precursors via the incomplete citric acid cycle; could
be an early evolution stage of the citric acid cycle.
Active site 1
Active site 2
The long flexible
arm of biotin
switches between
two active sites
of pyruvate
carboxylase
Incomplete citric
acid cycle has been
found in some
anaerobic bacteria
7. Net conversion of acetate to
carbohydrates is allowed via the
glyoxylate cycle
• 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,
found in plants, certain invertebrates, and some
microorganisms (including E. coli and yeast).
• The glyoxylate cycle shares three steps and bypasses
the rest, including the two decarboxylation steps, of
the citric acid cycle.
• Two acetyl-CoA molecules enter each glyoxylate
cycle with a net production of one succinate.
• Isocitrate lyase and malate synthase are the two
bypassing enzymes, converting isocitrate to malate
via the glyoxylate intermediate, releasing a succinate
on the way.
The glyoxylate
cycle
8. Conversion of fatty acids to glucose
(in germinating seeds) occurs in three
intracellular compartments
• Fatty acids are first degraded into acetyl-CoA,
which is in turn converted to succinate via the
glyoxylate cycle in glyoxysomes.
• Succinate is transported to mitochondria and is
converted to malate there via the citric acid cycle.
• Malate is then transported out of mitochondria and
is converted to glucose in the cytosol.
Fatty acids in germinating seeds are
converted to glucose via three compartments
9. The pyruvate dehydrogenase
complex in vertebrates is regulated
alloseterically and covalently
• The formation of acetyl-CoA from pyruvate is a
key irreversible step in animals because they are
unable to convert acetyl-CoA into glucose.
• The complex (in all organisms) is allosterically
inhibited by signaling molecules indicating a rich
source of energy, e.g., ATP, acetyl-CoA, NADH,
fatty acids; activated by molecules indicating a lack
(or demand) of energy, e.g., AMP, CoA, NAD+, Ca2+
• The activity of the complex (in vertebrates, probably
also in plants, but not in E. coli) is also regulated by
reversible phosphorylation of one of the enzymes,
E1, in the complex: phosphorylation of a specific Ser
residue inhibits and dephosphorylation activates the
complex.
• The kinase and phosphatase is also part of the
enzyme complex.
• The kinase is activated by a high concentration of
ATP.
10. The rate of the citric acid cycle is
controlled at three exergonic
irreversible steps
• Citrate synthase, isocitrate dehydrogenase and a-
ketoglutarate dehydrogenase;
• Inhibited by product feedback (citrate, succinyl-CoA)
and high energy charge (ATP, NADH);
• Activated by a low energy charge (ADP) or a signal
for energy requirement (Ca2+).
11. The partitioning of isocitrate,
between the citric acid and glyoxylate
cycles is coordinately regulated
• The activity of the E. coli isocitrate dehydrogenase
is inhibited when phosphorylated by a specific
kinase and activated when dephosphorylated by a
specific phosphatase.
• The kinase and phosphatase activities are located in
two domains of the same polypeptide and are
reciprocally regulated: the kinase is allosterically
inhibited (while the phosphatase activated) by
molecules indicating an energy depletion, e.g.,
accumulation of intermediates of glycolysis and
citric acid cycle.
• The allosteric inhibitors of the kinase also
act as inhibitors for the lyase: i.e., they
activate the dehydrogenase while
simultaneously inhibit the lyase.
The isocitrate
dehydrogenase and
the isocitrate lyase
are coordinately
regulated
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 drawn off to
synthesize many other biomolecules, including fatty
acids, steroids, amino acids, heme, pyrimidines, and
glucose.
• Oxaloacetate can get supplemented from pyruvate,
via a carboxylation reaction catalyzed by the biotincontaining 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 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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