Chapter 16

For Biochemistry II lecture of September 25, 2007

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).

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!

might be intermediates in
the intracellular oxidation
of pyruvate to CO2 and
H2O were searched in the
Citrate was found to be synthesized from oxaloacetate
1930s

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.

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 animal tissues. Enzymologica 4:148-156.

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

In 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 H+
gradient, which will promote ATP formation.

Mitochondria was found to be th

major site for fuel oxidation to
generate ATP.

transported into
mitochondria via a
specific transporter
on the inner
membrane and then
oxidized to acetylCoA by the catalysis
of pyruvate

The oxidative decarboxylation of

irreversible
pyruvateReaction
in mitochondria.
Stoichiometric
cofactors

Catalytic
cofactors

Coenzyme A was first discovered

by Lipmann in 1945.
A

Coenzyme A (CoA-SH) delivers

activated acyl groups (with 2-24
carbons) for degradation or

The arm
(~14 A)

Lipoate acts as both electron

and acyl carriers.
It swings between the three
different active sites of the
pyruvate dehydrogenase
Arsenic complex.
compounds are poisonous
because they sequester lipoamide.

The five reactions catalyzed by

pyruvate dehydrogenase complex:

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

The citric acid

cycle consists of
eight successive
reactions

The cycle begins with the

condensation of acetyl-CoA
and oxaloacetate to form
citrate
Aldol condensation
followed by a hydrolysis

( )

Hydrolysis of the thioester bond

releases a large amount of free energy.

( )

Citrate synthase before binding to oxaloacetate

Citrate synthase after binding to oxaloacetate

Acetyl-CoA analog

Active site is located at the interface of the two subunits.

A prochiral molecule

Aconitase contains
an iron-sulfur center

Aconitase then catalyzes

the interconversion
of citrate and isocitrate
via dehydration and
hydration.

Isocitrate is then converted

to -ketoglutarate via
oxidative decarboxylation,
producing CO2.

-
Oxalosuccinate is an intermediate; the carbon released as CO2 did
not come from the acetyl-CoA entering the cycle.

converted to succinyl-CoA via

another oxidative
decarboxylation, producing the
second CO2.
TPP

lipoate
FAD

converted to succinate,
accompanied by the
formation of a GTP (or
ATP)

Synthetase( ): ATP-consuming;
Synthase( ): not ATP-consuming.

mechanism for the

succinyl-CoA
synthetase
catalyzed GDP
phosphorylation,
via a
phosphoenzyme
Aintermediate.
phosphohistidine

An acyl phosphate

ATP

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!

The only substrate-level

phosphorylation in the cycle

Succinate is then converted to

fumarate via dehydrogenation

(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)

Malonate is a strong competitive

inhibitor of succinate dehydrogenase

Fumarate is then converted to

L-malate via hydration

A stereospecific enzyme only

acting on the trans and L isomers

Neither maleate nor D-malate

are substrates of fumarase

The cycle ends by the regeneration of

oxaloacetate from L-malate

None of the intermediates

are phosphorylated.

All are either di- or

tricarboxylic acids.

An overview of
the citric acid cycle
3

To regenerate
oxaloacetate.

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!

The symmetric molecule

can react asymmetrically
in the asymmetric active
site of enzymes.

Citrate is a prochiral molecule

Active site of aconitase

Overall efficiency
of energy conservation:

65%

Accumulating evidence also

suggest the formation of
multienzyme complexes (or
metabolons) for the citric
acid cycle enzymes,
facilitating substrate
channeling between the
active sites.

The citric acid

intermediates are
important sources for
biosynthetic precursors.

The citric acid cycle

is amphibolic.

The citric acid cycle intermediates

need to be replenished
Activated by acetyl-CoA

Activated by Fru-1,6P

A proposed mechanism for pyruvate carboxylase:

biotin acts as a tethered long arm that carries the
activated CO2.

Common biological tethers found in proteins

-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!

Activity of pyruvate
dehydrogenase
complex is regulated
by allosteric and
covalent
mechanisms.

Phosphorylated
E1 is inactive.

Inhibits PFK-1

nzymes are switched of

hen the energy charge is
high and biosynthetic
ermediates are abundant.

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 leve
of ATP for the cells.

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. 988991

The glyoxylate
cycle

The two decarboxylation

steps of the citric acid
cycle are bypassed.

Partial overlapping
of the glyoxylate and
citric acid cycles

Conversion of fatty acids to glucose (in

germinating seeds) occurs in three
intracellular compartments

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.

 Pyruvate

Summary

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 biotin-containing 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.