Transcript The Chemical Logic of TCA cycle

Chapter 19
The Tricarboxylic Acid Cycle
Biochemistry
by
Reginald Garrett and Charles Grisham
Essential Question
 How is pyruvate oxidized under aerobic
conditions
Pyruvate from glycolysis is converted to acetyl-CoA
and oxidized to CO2 in the tricarboxylic acid (TCA)
cycle
 What is the chemical logic that dictates how this
process occurs?
Hans Krebs showed that the oxidation of
acetate is accomplished by a cycle
TCA cycle, Citric Acid Cycle or Krebs Cycle
• Pyruvate from glycolysis is oxidatively
decarboxylated to acetate and then degraded to
CO2 in TCA cycle
• Some ATP is produced
• More NADH and FADH2 are made (24 electrons)
• NADH and FADH2 go on to make more ATP in
electron transport and oxidative phosphorylation
(chapter20)
Figure 19.1 (a) Pyruvate produced in glycolysis is oxidized in (b) the tricarboxylic acid (TCA)
cycle. (c) Electrons liberated in this oxidation flow through the electron-transport chain and
drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall
process occurs in mitochondria.
19.1 – What Is the Chemical Logic of the
TCA Cycle?
•
TCA cycle seems like a complicated way to
oxidize acetate units to CO2
• Normal ways to cleave C-C bonds in
biological systems:
1. cleavage between Carbons  and  to a
carbonyl group (-cleavage)
O
(fructose bisphosphate aldolase)
— C—C— C—
2. -cleavage of an -hydroxyketone
(transketolase; fig 22.31)
O OH
— C—C—
The Chemical Logic of TCA cycle
• Neither of these cleavage strategies is
suitable for acetate
• Living things have evolved the clever
chemistry of condensing acetate with
oxaloacetate and carry out a -cleavage.
• TCA combines this -cleavage reaction
with oxidation to form CO2, regenerate
oxaloacetate and capture all the energy in
NADH and ATP
Figure 19.2
The tricarboxylic acid cycle.
19.2 – How Is Pyruvate Oxidatively
Decarboxylated to Acetyl-CoA?
• Pyruvate must enter the mitochondria to enter
the TCA cycle
• Oxidative decarboxylation of pyruvate is
catalyzed by the pyruvate dehyrogenase
complex
Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH + H+
• Pyruvate dehydrogenase complex is a
noncovalent assembly of three enzymes
• Five coenzymes are required
Pyruvate dehydrogenase complex (PDC):
• Three enzymes and five coenzymes:
E1: pyruvate dehydrogenase (24)
thiamine pyrophosphate
E2: dihydrolipoyl transacetylase (24)
lipoic acid
E3: dihydrolipoyl dehydrogenase (12)
FAD
NAD+
CoA
• The product of the first enzyme is pass
directly to the secondenzyme
(a) Domain structure of E2 and E3BP subunits
(b) a truncated version of E2
L1
L2
E1BD
IC
L3
E3BD
30 E1 &
12 E3
(c) Model of the E2/E3BP:E3 core complex
(6 E3BP dimer & 48 E2)
(d) Model of human PDC
Figure 19.3 Models of human pyruvate dehydrogenase
Figure 19.4 The reaction mechanism of the pyruvate
dehydrogenase complex
(TPP)
Figure 19.5 The mechanism of the first three steps of the pyruvate
dehydrogenase complex reaction
The Coenzymes of the Pyruvate Dehydrogenase
Complex
Thiamine pyrophosphate (vitamin B1 analog)
TPP assists in the decarboxylation of α-keto acids (here) and in
the formation and cleavage of α-hydroxy ketones (as in the
transketolase reaction; see Chapter 22).
The Nicotinamide Coenzymes (vitamin B3, niacin analog)
NAD+/NADH and
NADP+/NADPH carry out
hydride (H:-) transfer reactions.
All reactions involving these
coenzymes are two-electron
transfers.
The Flavin Coenzymes (vitamin B2)
FAD/FADH2
Flavin coenzymes can exist
in any of three oxidation
states, and this allows flavin
coenzymes to participate in
one-electron and twoelectron transfer reactions.
Partly because of this,
flavoproteins catalyze many
reactions in biological
systems and work with
many electron donors and
acceptors.
Coenzyme A (vitamin B5, pantothenic acid)
The two main functions of Co A are:
1. Activation of acyl groups for transfer
by nucleophilic attack
2. Activation of the α-hydrogen of the
acyl group for abstraction as a proton
• The reactive sulfhydryl group on CoA
mediates both of these functions.
• The sulfhydryl group forms thioester
linkages with acyl groups.
• The two main functions of CoA are
illustrated in the citrate synthase
reaction (see Figure 19.6).
Lipoic Acid
1. Lipoic Acid functions to couple acyl-group transfer and electron
transfer during oxidation and decarboxylation of α-keto acids.
2. It is found in pyruvate dehydrogenase and α-ketoglutarate
dehydrogenase.
3. Lipoic acid is covalently bound to relevant enzymes through
amide bond formation with the ε-NH2 group of a lysine side
chain.
19.3 – How Are Two CO2 Molecules
Produced from Acetyl-CoA?
Tricarboxylic acid cycle, Citric acid cycle,
and Krebs cycle
• Pyruvate is oxidatively decarboxylated to form
acetyl-CoA
Citrate (6C)→ Isocitrate (6C)→ -Ketoglutarate (5C) →
Succinyl-CoA (4C) → Succinate (4C) → Fumarate (4C)
→ Malate (4C) → Oxaloacetate (4C)
1. Citrate synthase reaction
• Acetyl-CoA reacts with oxaloacetate in a Perkin
condensation (A carbon-carbon condensation between a
ketone or aldehyde and an ester)
Figure 19.6 Citrate is formed in the citrate synthase reaction from oxaloacetate
and acetyl-CoA. The mechanism involves nucleophilic attack by the carbanion of
acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester
hydrolysis.
• Citrate synthase
– is a dimer
– NADH & succinyl-CoA are
allosteric inhibitors
• Large, negative G -irreversible
Figure 19.7 Citrate synthase in mammals is a
dimer of 49-kD subunits. In the monomer
shown here, citrate (blue) and CoA (red) bind to
the active site, which lies in a cleft between two
domains and is surrounded mainly by α-helical
segments.
2. Citrate Is Isomerized by Aconitase to
Form Isocitrate
• Citrate is a poor substrate for oxidation because
it contains a tertiary alcohol
• So aconitase isomerizes citrate to yield isocitrate
which has a secondary -OH, which can be
oxidized
• Note the stereochemistry of the reaction:
aconitase removes the pro-R H of the pro-R arm
of citrate
• Aconitase uses an iron-sulfur cluster (Fig. 19.9)
Aconitase Utilizes an Iron-Sulfur Cluster
• Fluoroacetate is an extremely poisonous agent
that blocks the TCA cycle
• Rodent poison: LD50 is 0.2 mg/kg body
weight
• Aconitase inhibitor
3. Isocitrate Dehydrogenase Catalyzes the
First Oxidative Decarboxylation in the Cycle
•
Catalyzes the first oxidative decarboxylation
in the cycle
1. Oxidation of C-2 alcohol of
isocitrate with concomitant
reduction of NAD+ to NADH
2. followed by a -decarboxylation
reaction that expels the central
carboxyl group as CO2
Isocitrate Dehydrogenase
•
•
Isocitrate dehydrogenase links the TCA cycle
and electron transport pathway because it
makes NADH
Isocitrate dehydrogenase is a regulation
reaction
–
–
•
NADH and ATP are allosteric inhibitor
ADP acts as an allosteric activator
-ketoglutarate is also a crucial -keto acid for
aminotransferase reactions (Chapter 25),
connecting the TCA cycle (carbon metabolism)
with nitrogen metabolism
4. -Ketoglutarate Dehydrogenase
•
•
Catalyzes the second oxidative decarboxylation
of the TCA cycle
This enzyme is nearly identical to pyruvate
dehydrogenase - structurally and
mechanistically
1. -ketoglutarate dehydrogenase
2. Dihydrolipoyl transsuccinylase
3. Dihydrolipoyl dehydrogenase (identical to PDC)
•
Five coenzymes used - TPP, CoA-SH, Lipoic
acid, NAD+, FAD
Like pyruvate dehydrogenase, -ketoglutarate dehydrogenase is a
multienzyme complex – consisting of -ketoglutarate dehydrogenase,
dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase.
The complex uses five different coenzymes.
19.4 – How Is Oxaloacetate Regenerated
to Complete the TCA Cycle?
5. Succinyl-CoA Synthetase
A substrate-level phosphorylation
GTP + ADP
ATP + GDP
(nucleotide diphosphate kinase)
• A nucleoside
triphosphate is made
Thioester
• Its synthesis is driven by
hydrolysis of a CoA ester
[Succinyl-P]
• The mechanism involves
a phosphohistidine
[Phosphohistidine]
GTP
Figure 19.11 The mechanism of the
succinyl-CoA synthetase reaction.
Completion of the TCA Cycle –
Oxidation of Succinate to Oxaloacetate
• This process involves a series of three reactions
• These reactions include:
1. Oxidation of a single bond to a double bond
(FAD/FADH2)
2. Hydration across the double bond
3. Oxidation of the resulting alcohol to a ketone
(NAD+/NADH)
• These reactions will be seen again in oxidation of fatty acids
6. Succinate Dehydrogenase
• The oxidation of succinate to fumarate
• A membrane-bound enzyme is actually part
of the electron transport chain in the inner
mitochondrial membrane (succinate-CoQ
reductase)
• The reaction is not sufficiently exergonic to
reduce NAD+
(trans-)
• The electrons transferred from succinate to FAD (to
form FADH2) are passed directly to ubiquinone (UQ)
in the electron transport pathway (chapter 20)
• FAD is covalently bound to the enzyme
• Contains iron-sulfur cluster
Succinate Dehydrogenase
contains three types of ironsulfur clusters: a 4Fe-4S
cluster, a 3Fe-4S cluster, and
a 2Fe-2S cluster.
Figure 19.12 The covalent bond
between FAD and succinate
dehydrogenase links the C-8a
carbon of FAD and the N-3 of
a His residue of the enzyme.
7. Fumarase
Hydration across the double bond
• Catalyzes the trans-hydration of fumarate to
form L-malate
• trans-addition of the elements of water across
the double bond
• Possible mechanisms are shown in Figure 19.13
8. Malate Dehydrogenase
• Completes the Cycle by Oxidizing L-Malate to
Oxaloacetate
• This reaction is very endergonic, with a Go' of
+30 kJ/mol
19.5 – What Are the Energetic Consequences
of the TCA Cycle?
One acetate through the cycle produces two CO2,
one ATP, four reduced coenzymes
Acetyl-CoA + 3 NAD+ + FAD + ADP + Pi + 2 H2O →
2 CO2 + 3 NADH + 3 H+ + FADH2 + ATP + CoASH
G0’ = -40kJ/mol
Glucose + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi + 2 H2O →
6 CO2 + 10 NADH + 10 H+ + 2 FADH2 + 4 ATP
NADH + H+ + 1/2 O2 + 3 ADP + 3 Pi → NAD+ + 3ATP + H2O
FADH2 + 1/2 O2 + 2 ADP + 2 Pi → FAD + 2ATP + H2O
The Carbon Atoms of Acetyl-CoA Have
Different Fates in the TCA Cycle
Neither of the carbon atoms of a labeled acetate
unit is lost as CO2 in the first turn of the cycle
1.Carbonyl C of acetyl-CoA turns to CO2 only
in the second turn of the cycle (following entry
of acetyl-CoA )
2.Methyl C of acetyl-CoA survives two cycles
completely, but half of what's left exits the
cycle on each turn after that.
The Carbon Atoms of Acetyl-CoA Have Different
Fates in the TCA Cycle
Figure 19.15
The fate of the carbon atoms of acetate in successive TCA cycles. (a) The carbonyl carbon of
acetyl-CoA is fully retained through one turn of the cycle but is lost completely in a second
turn of the cycle.
19.6 – Can the TCA Cycle Provide
Intermediates for Biosynthesis?
The products in TCA cycle also fuel a
variety of biosynthetic processes
• α-Ketoglutarate is transaminated to make
glutamate, which can be used to make purine
nucleotides, Arg and Pro
• Succinyl-CoA can be used to make porphyrins
• Fumarate and oxaloacetate can be used to make
several amino acids and also pyrimidine
nucleotides
Figure 19.16
The TCA cycle
provides
intermediates
for numerous
biosynthetic
processes in
the cell.
• Citrate can be exported from the mitochondria
and then broken down by citric lyase to yield
acetyl-CoA and oxaloacetate (chapter 24)
• Oxaloacetate is rapidly reduced to malate
• Malate can be transported into mitochondria
or oxidatively decarboxylated to pyruvate by
malic enzyme
• Oxaloacetate can also be decarboxylated to
yield phosphoenolpyruvate
19.7 – What Are the Anaplerotic, or
“Filling Up,” Reactions?
• Pyruvate carboxylase - converts pyruvate to
oxaloacetate (in animals), is activated by
acetyl-CoA (chapter 22, gluconeogenesis)
• PEP carboxylase - converts PEP to
oxaloacetate (in bacteria & plants), inhibited
by aspartate
• Malic enzyme converts pyruvate into malate
• The catabolism of amino acids provides
pyruvate, acetyl-CoA, oxaloacetate, fumarate,
-ketoglutarate, and succinate (chapter 25).
• PEP carboxykinase
– Could have been an anaplerotic reaction.
– CO2 binds weakly to the enzyme, whereas
oxaloacetate binds tightly
– The reaction favors formation of PEP from
oxaloacetate
19.8 – How Is the TCA Cycle Regulated?
1. Citrate synthase
– ATP, NADH and succinyl-CoA inhibit
2. Isocitrate dehydrogenase
– ATP and NADH inhibits
– ADP and NAD+ activate
3.  -Ketoglutarate dehydrogenase
– NADH and succinyl-CoA inhibit
– AMP activates
4. Pyruvate dehydrogenase
– ATP, NADH, acetyl-CoA inhibit
– NAD+, CoA activate
Regulation of the TCA cycle.
19.8 – How Is the TCA Cycle Regulated?
When the ADP/ATP or NAD+/NADH ratio
is high, the TCA cycle is turned on
Succinyl-CoA is an intracycle regulator,
inhibiting citrate synthase and ketoglutarate dehydrogenase
Acetyl-CoA acts as a signal to the TCA
cycle that glycolysis and fatty acid breakdown is producing two-carbon unit
1. Activate pyruvate carboxylase
2. Feedback inhibit pyruvate dehydrogenase
Pyruvate dehydrogenase is regulated by
phosphorylation/dephosphorylation
Animals cannot synthesize glucose from acetylCoA, so pyruvate dehydrogenase complex plays
a pivotal role in metabolism
• Allosterically regulation
– Inhibit by Acetyl-CoA (dihydrolipoyl transacetylase), or
NADH (dihydrolipoyl dehydrogenase)
• Covalently modification on pyruvate dehydrogenase
– Phosphorylation (pyruvate dehydrogenase kinase)
– Dephosphorylation (pyruvate dehydrogenase phosphatase)
1. The pyruvate dehydrogenase kinase (PDK; Fig 19.3)
is associated with the enzyme
– Allosterically activated by NADH and acetyl-CoA
– Phosphorylated pyruvate dehydrogenase subunit is
inactive
2. Reactivation of the enzyme by
pyruvate dehydrogenase phosphatase
– A Ca2+-activated enzyme
– Hydrolyzes the phosphoserine
moiety on the dehydrogenase
subunit
– Insulin and Ca2+ activate
dephosphorylation
– Pyruvate inhibit dephosphorylation
19.9 – Can Any Organisms Use Acetate as
Their Sole Carbon Source?
• Plant and some bacteria can use acetate as the
only source of carbon for all the carbon
compounds
• plants and some bacteria employ a modification
of the TCA cycle called the glyoxylate cycle to
produce four-carbon compounds from acetylCoA
• The CO2-producting steps are bypassed and an
extra acetate is utilized
• Isocitrate lyase and malate synthase are the
short-circuiting enzymes (Fig 19.21)
Figure 19.21 The glyoxylate cycle.
Glyoxylate Cycle
In plants, the glyoxylate cycle is carried out in
glyoxysomes, but yeast and algae carry out in
cytoplasm
1.Isocitrate lyase
– produces glyoxylate and succinate
– Is similar to the aldolase reaction in glycolysis
2.Malate synthase
– A Claisen condensation of acetyl-CoA and the
aldehyde group of glyoxylate to form L-malate
– Is similar to the citrate synthase reaction
Figure 19.22 The isocitrate lyase reaction.
•
The glyoxylate cycle helps plants grow in the
dark
–
–
–
•
Certain seeds grow underground, where
photosynthesis is impossible
Many seeds are rich in lipids
Once the growing plant begins photosynthesis and
can fix CO2 to produce carbohydrate, the
glyoxysomes disappear
Glyoxysomes must borrow three reactions
from mitochondria: succinate to oxaloacetate
1. Succinate dehydrogenase
2. Fumarate
3. Malate dehydrogenase
Figure 19.23
Glyoxysomes lack three of
the enzymes needed to
run the glyoxylate cycle.
Succinate dehydrogenase,
fumarase, and malate
dehydrogenase are all
“borrowed” from the
mitochondria in a shuttle
in which succinate and
glutamate are passed to
the mitochondria, and
α-ketoglutarate and
aspartate are passed to
the glyoxysome.