Chapter 16

The Citric Acid Cycle

The common pathway leading to the
complete oxidation of carbohydrates,
fatty acids, and amino acids to CO2
A pathway providing many precursors
for biosynthetic reactions
 Citric Acid Cycle ( 柠檬酸循环 )
• Also called the tricarboxylic acid cycle (TCA)
or the Krebs cycle
• Pyruvate completely oxidized to CO 2 and H2O in
the presence of O2---cellular respiration
• Occurs in eight steps in mitochondria
• Energy efficiently conserved
• A hub in metabolism, serving in both catabolic
and anabolic processes
 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 Acetyl-CoA is completely oxidized to
CO2, with electrons collected by NAD and FAD
via the citric acid cycle.
• Stage III Electrons of NADH and FADH2 are
transferred to O2 via a series of carriers,
producing H2O and a H+ gradient, which will
promote ATP formation.
 2. Pyruvate is oxidized to
acetyl-CoA by the pyruvate
dehydrogenase complex
• Pyruvate is first transported into mitochondria
via a specific transporter on the inner membrane.
• Pyruvate is converted to acetyl-CoA and CO 2 by
oxidative decarboxylation.
• The pyruvate dehydrogenase (PDH) complex is a
huge multimeric assembly of three kinds of
enzymes, having 60 subunits in bacteria and
more in mammals.
Production of acetyl-CoA

irreversible

Oxidative decarboxylation
Critical to its role
as an acyl carrier
in a number of
metabolic reactions
(thioester)
Lipoate can act as
a carrier of both
hydrogen and
an acetyl group
Pyruvate dehydrogenase complex (PDH)

Three-enzyme complex
E1: pyruvate dehydrogenase (TPP)
E2: dihydrolipoyl transacetylase (lipoate)--core
E3: dihydrolipoyl dehydrogenase (FAD)

Plus 2 regulatory proteins (a protein kinase

and a phosphoprotein phosphatase)
Cryoelectron
micrograph of PDH
complexes isolated
from bovine kidney
Three-dimensional image of the PDH complex
E2 consists of three types of domains linked
by short polypeptide linkers
Oxidative decarboxylation of pyruvate to
acetyl-CoA by the PDH complex
• Substrates of the five reactions catalyzed by the
pyruvate dehydrogenase complex are efficiently
channeled---substrate channeling
• The long lipoyllysine arm of E2 swings from the
active site of E1 to E2 to E3, tethering the
intermediates to the enzyme complex to allow
substrate channeling.

• The multienzyme complexes catalyzing the oxidative

decarboxylation of a few different kinds of a-keto
acids, including pyruvate dehydrogenase complex,
a-ketoglutarate dehydrogenase complex and
branched-chain a-keto acid dehydrogenase complex,
show remarkable structure and function similarities
(all have identical E3, similar E1 and E2).
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 (-
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.
• 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).
 The Nobel Prize in Physiology
or Medicine 1953
“for his discovery of the citric acid cycle”

Sir Hans Adolf Krebs

Great Britain

Sheffield University,
Sheffield,
Great Britain

1900-1981
 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.
Reactions of the citric acid cycle
Step 1: condensation

irreversible
Structure of citrate synthase
Citrate synthase
---induced fit
Step 2: dehydration & hydration
Step 3 oxidative decarboxylation

irreversible

Two isozymes
Step 4 oxidative decarboxylation

irreversible

Similar to pyruvate dehydrogenase complex

A conserved mechanism for oxidative decarboxylation
Step 5 substrate-level phosphorylation

(Two isozymes, one for

GDP, the other for ADP)
Step 6 dehydrogenation

(Membrane-bound)
Competitive Inhibitor
 Step 7
hydration

Stereospecific
(trans) (cis)
Step 8 dehydrogenation
Reactions of the citric acid cycle
Products of one turn of the citric acid cycle
Products of one turn of the citric acid cycle
 irreversible

Reactions of the citric acid cycle

 The citric acid cycle

Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O

2CO2 + 3NADH + FADH2 + GTP + 3H+ + CoA

 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.
• The 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 intermediates of the citric
acid cycle 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).
• Any compounds that give rise to four- or five-
carbon intermediates of the cycle can be oxidized
by the cycle.
• It provides precursors for the biosynthesis of
glucose, amino acids, nucleotides, fatty acids,
heme groups, etc.
Citric acid cycle provides precursors for many
biosynthetic pathways
• 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 (a vitamin) as the coenzyme.
• 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

pyruvate carboxylase.
Biotin (a vitamin)
plays a key role in
the carboxylation
reaction---
specialized
carrier of
one-carbon groups
Some anaerobic bacteria,
lacking -ketoglutarate
dehydrogenase, make
biosynthetic precursors
via the incomplete citric acid
cycle, which could be an
early evolution stage of
the citric acid cycle.
Regulation of the citric acid cycle
Regulatory enzymes:
Pyruvate dehydrogenase complex
Citrate synthase
Isocitrate dehydrogenase
a-ketoglutarate dehydrogenase
 7. The pyruvate dehydrogenase
complex in vertebrates is regulated
allosterically and covalently
• The formation of acetyl-CoA from pyruvate is a
key irreversible step in animals.
• The complex (in all organisms) is allosterically
inhibited by signaling molecules indicating a rich
source of energy, e.g., ATP, acetyl-CoA, NADH,
long chain 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.
E1 (unphosphorylated)---active
E1 (phosphorylated)---inactive
Kinase involved is activated by ATP,
but inhibited by a drug called
dichloroacetate.
 8. The rate of the citric acid cycle is
controlled at three exergonic
irreversible steps
• Regulatory enzymes: citrate synthase, isocitrate
dehydrogenase and -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+).
Regulation of the citric acid cycle *****
 9. Net conversion of acetate to
carbohydrates is achieved via
the glyoxylate cycle
• In vertebrates 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
• 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
Convert acetate to carbohydrate in plants,
certain invertebrates and some
microorganisms

2 Acetyl-CoA + NAD+ + 2H2O

Succinate + 2CoA + NADH + H+

Electron micrograph of a germinating cucumber seed
 10. Conversion of fatty acids to
glucose (in germinating seeds) occurs
in three intracellular compartments
• Fatty acids are first converted to acetyl-CoA,
which is in turn converted to succinate via the
glyoxylate cycle in glyoxysomes.
• Succinate is transported to mitochondria and
converted to malate via the citric acid cycle.
• Malate is then transported out of mitochondria
and is converted to glucose in the cytosol.
Relationship between the citric acid cycle and the glyoxylate cycle
11. The partitioning of isocitrate between
the citric acid cycle and the glyoxylate
cycle 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 citric acid
and glyoxylate
cycles are
coordinately
regulated
• 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 the citric acid
cycle.

• The allosteric inhibitors of the kinase also act

as inhibitors for the isocitrate lyase: i.e., they
activate the dehydrogenase while
simultaneously inhibit the lyase.
 Keywords
• Pyruvate dehydrogenase complex--reaction, regulation
• The citric acid cycle--reaction, regulation
• The glyoxylate cycle--products
 Words of the week
• kinase vs. phosphatase
• phosphorylation vs. dephosphorylation
• phosphorylated vs. unphosphorylated
• activated vs. inactivated
• synthase vs. synthetase
 Textbook reading of the week
• Summary 16.1 (p637)
• Summary 16.2 (p653)
• Summary 16.3 (p656)
• Summary 16.4 (p659)
• p646 (Box 16-2)
• p648 (Box 16-3: Citrate: a symmetric molecule
that reacts asymmetrically)
• p653-655 (regulation of the citric acid cycle)
 Summary
• Pyruvate, the product of glycolysis, is converted to
acetyl-CoA, the starting material of the citric acid
cycle, by the pyruvate dehydrogenase (PDH) complex.

• The PDH complex is composed of multiple copies of

three enzymes and five different coenzymes.

• The citric acid cycle is a nearly universal central

catabolic pathway in which compounds derived from
the breakdown of carbohydrates, fats and proteins are
oxidized to CO2, with most of the energy of oxidation
temporarily held in the electron carriers NADH and
FADH2.
• For each acetyl-CoA oxidized by the citric acid cycle,
the energy gain consists of three molecules of NADH,
one FADH2, and one ATP or GTP.

• The overall rate of the citric acid cycle is controlled

by the rate of conversion of pyruvate to acetyl-CoA
and by the flux through citrate synthase, isocitrate
dehydrogenase, and -ketoglutarate dehydrogenase.

• The PDH complex is inhibited allosterically by

metabolites that signal a sufficiency of metabolic
energy (ATP, acetyl-CoA, NADH and fatty acids) and
stimulated by metabolites that indicate a reduced
energy supply (AMP, NAD+ and CoA).
• The glyoxylate cycle is active in the germinating seeds
of some plants and in certain microorganisms that
can live on acetate as the sole carbon source.
• In the glyoxylate cycle, the bypassing of the two
decarboxylation steps of the citric acid cycle makes
possible of the net formation of succinate.
• Vertebrates lack the glyoxylate cycle and cannot
synthesize glucose from acetate or the fatty acids that
give rise to acetyl-CoA.

• The partitioning of isocitrate between the citrate acid

cycle and the glyoxylate cycle is controlled at the level
of isocitrate dehydrogenase, which is regulated by
reversible phosphorylation.