Chapter 21 Citric acid cycle

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Chapter 21 Citric acid cycle
Learn to draw the TCA cycle, including the names
and structures of intermediates and the names of
the enzymes.
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Citric acid cycle aka tricarboxylic acid (TCA)
cycle or Krebs cycle

• Glycolysis - cytosol
• Fatty Acid Oxidation Mitochondrial matrix
• Citric Acid Cycle Mitochondrial matrix
• Oxidative Phosphorylation Inner membrane of
  mitochondria

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After Glycolysis
What next
Glucose 2 ADP 2 Pi 2 NAD
Glycolysis
2 Pyruvate 2 ATP 2 NADH 2 H 2 H2O

• Cell needs to regenerate the NAD that was
  utilized in glycolysis
• NAD regeneration can be accomplished by
  aerobic or anaerobic metabolism

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Three possible fates of pyruvate
Aerobic Metabolism
Pyruvate
NADH H
Anaerobic metabolism
Lactate dehydrogenase
Acetaldehyde
NAD
Acetyl CoA
NADH H
Alcohol dehydrogenase
L-lactate
NAD
Homolactic fermentation
TCA cycle
Ethanol
Alcoholic fermentation
Electron transport
Adapted from Chemistry 153A UCLA Course
Compendium by M. A. Bates
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Glycolysis does not require O2 from air, but it
does require the oxidant NAD
Ethanol fermentation in yeast and other
microorganisms
Lactic acid fermentation in several types of
animal cells and some microorganisms
From Lehninger Principles of Biochemistry
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Thiamine pyrophosphate (TPP)
From Lehninger Principles of Biochemistry
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Mechanism of Pyruvate Decarboxylase
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Aerobic Metabolism
Pyruvate dehydrogenase complex
A cluster of 3 enzymes located in the
mitochondria of eukaryotic cells and cytosol of
prokaryotes A classic well studied example of a
multienzyme complex
Non-covalent stable aggregate of enzymes
From Lehninger Principles of Biochemistry
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Ethanol fermentation in yeast and other
microorganisms
From Lehninger Principles of Biochemistry
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Overall reaction catalyzed by the pyruvate
dehydrogenase complex
Oxidative decarboxylation
Irreversible reaction Requires sequential action
of - 3 different enzymes 5 different
coenzymes
From Lehninger Principles of Biochemistry
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• Different Coenzymes or Prosthetic Groups
  Required for Pyruvate Dehydrogenase Complex are
• Thiamine pyrophosphate (TPP)
• Flavin adenine dinucleotide (FAD)
• Coenzyme A (CoA)
• Nicotinamide adenine dinucleotide (NAD)
• Lipoate

Four different vitamins required in human
nutrition are vital components of this system
Thiamine (in TPP) Riboflavin (in FAD) Niacin
(in NAD) Pantothenate (in CoA)
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• Thiamine pyrophosphate (TPP)

From Lehninger Principles of Biochemistry
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2. Flavin adenine dinucleotide (FAD)
From Lehninger Principles of Biochemistry
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3. Coenzyme A (CoA)
Critical to its role as an acyl carrier
(vitamin B5)
Hydroxyl group of pantothenic acid is joined to
ADP moeity
Modified ADP
Carboxylic group attached to b-mercaptoethylamine
From Lehninger Principles of Biochemistry
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4. Nicotinamide adenine dinucleotide (NAD)
From Lehninger Principles of Biochemistry
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5. Lipoate
Lipoate has 2 thiol groups
Lipoyllysyl moiety is the prosthetic group of
dihydrolipoyl transacetylase (E2)
From Lehninger Principles of Biochemistry
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Pyruvate Dehydrogenase Complex

• Consists of 3 distinct enzymes
• 1) Pyruvate dehydrogenase (E1)
• 2) Dihydrolipoyl transacetylase (E2)
• 3) Dihydrolipoyl dehydrogenase (E3)
• The 3 enzymes are bound together in stable,
  non-covalent aggregates.
• Each enzyme is present in multiple copies
  within each aggregate.
• The of copies of each enzyme the actual size
  of the complex varies among organisms.

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Pyruvate Dehydrogenase Complex from E. coli

• E2 forms the core of the cluster to which
• other enzymes are attached
• E2 24 copies Contains bound lipoate
• E1 24 copies Contains bound TPP
• E3 12 copies Contains bound FAD

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Overall reaction catalyzed by the pyruvate
dehydrogenase complex
Oxidative decarboxylation
Irreversible reaction Requires sequential action
of - 3 different enzymes 5 different
coenzymes
From Lehninger Principles of Biochemistry
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Mechanism of Pyruvate Decarboxylase
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Enzyme Reaction Catalyzed E1 Reaction 1
2 E2 Reaction 3 E3 Reaction 4 5
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• Aerobic oxidation of glucose that includes
• the oxidation of pyruvate provides
• energy in the brain
• Thiamine (vitamin B1) deficiency
• - Unable to oxidize pyruvate normally
• Beriberi, a disease that is characterized by
• loss of neural function is due to thiamine
• deficiency

Habitual consumption of large amounts of alcohol
can lead to thiamine deficiency
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Beriberi - caused by a deficiency of thiamine
(vitamin B1) - affects the muscles, heart,
nerves, and digestive system - literally means
"I can't, I can't" in Singhalese, which reflects
the crippling effect it has on its victims -
common in parts of the developing world, where
white rice is the main food
www.nlm.nih.gov/medlineplus
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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Reactions of the citric acid cycle
Steps 1, 3, 4 irreversible
4 out of 8 steps are oxidations
Energy is conserved by electron transfer to FAD
or NAD, forming FADH2 or NADH H
All of these reactions take place in mitochondria
From Lehninger Principles of Biochemistry
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The TCA Cycle

• aka Citric Acid Cycle, Krebs Cycle
• Pyruvate (actually acetate) from glycolysis is
  degraded to CO2
• Some ATP is produced
• More NADH is made
• NADH goes on to make more ATP in electron
  transport and oxidative phosphorylation

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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Step 1 Formation of citrate
Condensation of acetyl CoA with oxaloacetate
1
2
S-CoA
From Lehninger Principles of Biochemistry
intermediate
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From Garrett Grisham
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Citrate synthase
Oxaloacetate
A stable analog of acetyl-CoA bound
Open form of enzyme
Closed form with oxaloacetate
From Lehninger Principles of Biochemistry
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Dimeric protein with 2 domains
Open form of enzyme
Closed form with oxaloacetate
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Step 2 Formation of Isocitrate via cis-Aconitate
The equilibrium mixture at pH 7.4 25 oC
contains less than 10 isocitrate In the
cell the reaction is pulled to the right because
isocitrate is consumed rapidly, lowering its
steady state concentration
From Lehninger Principles of Biochemistry
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Aconitase

• Isomerization of Citrate to Isocitrate
• Citrate is a poor substrate for oxidation
• So aconitase isomerizes citrate to yield
  isocitrate which has a secondary -OH, which can
  be oxidized
• Note the stereochemistry of the Rxn aconitase
  removes the pro-R H of the pro-R arm of citrate!
• Aconitase uses an iron-sulfur cluster

From Garrett Grisham
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Iron-sulfur center in aconitase
4
3
1
2
Basic residue (keeps the citrate in active site)
From Lehninger Principles of Biochemistry
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Aconitase
Fe
S
Fe
S
4Fe-4S cluster
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Proteins that contain iron-sulfur clusters play
an important role in biological systems
Rieske iron-sulfur proteins 2Fe-2S
Aconitase family 4Fe- 4S cluster
3Fe-4S cluster
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Functions of iron-sulfur proteins ( thus of
iron- sulfur clusters)

• when first discovered, they were assumed to
  function exclusively as electron donors and
  acceptors
• they have since been discovered in enzymes
  which control gene expression, the activity of
  other proteins, several other functions

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Inhibitors / Inactivators of Aconitase
Found in some plant leaves
Radicals such as superoxide (O2.-) can pull iron
out of the 4Fe-4S center of aconitase making the
enzyme inactive
Adapted from Garrett Grisham
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Step 3 Oxidation of Isocitrate
Oxidative decarboxylation
There are 2 different forms of isocitrate
dehydrogenase One requires NAD the other NADP
From Lehninger Principles of Biochemistry
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Isocitrate Dehydrogenase
Cofactor (can also use Mg2)

• Oxidative decarboxylation of isocitrate to yield
  ? -ketoglutarate
• Classic NAD chemistry
• - hydride removal followed by a
  decarboxylation
• Isocitrate dehydrogenase is a link to the
  electron transport pathway because it makes NADH

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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Step 4 Oxidation of a-Ketoglutarate to
Succinyl-CoA and CO2
? -Ketoglutarate Dehydrogenase

• A second oxidative decarboxylation
• This enzyme is nearly identical to pyruvate
  dehydrogenase
• - structurally and mechanistically
• Five coenzymes used - TPP, CoASH, Lipoic acid,
  NAD, FAD
• You know the mechanism if you remember pyruvate
  dehydrogenase

From Lehninger Principles of Biochemistry
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Pyruvate dehydrogenase complex
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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Step 5 Conversion of Succinyl-CoA to Succinate
Succinyl-CoA Synthetase

• A substrate-level phosphorylation
• A nucleoside triphosphate is made
• Its synthesis is driven by hydrolysis of a CoA
  ester
• The mechanism involves a phosphohistidine

From Lehninger Principles of Biochemistry
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ATP and GTP are rapidly interconverted through
the action of nucleosidediphosphate kinase
GTP ADP GDP ATP DGo
0
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The Succinyl-CoA Synthetase Reaction
From Lehninger Principles of Biochemistry
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Succinyl-CoA Synthetase from E. coli
partial positive charges of power helices in
dark blue and dark brown stabilize
phosphohistidyl group.
From Lehninger Principles of Biochemistry
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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Step 6 Oxidation of Succinate to Fumarate
Only membrane bound enzyme in the TCA cycle
Succinate dehydrogenase 1) Catalyzes the
stereospecific dehydrogenation of succinate to
fumarate 2) Contains FAD as a prosthetic group
(covalently linked to the enzyme via a His
residue)
From Lehninger Principles of Biochemistry
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Malonate is a strong competitive inhibitor of
succinate dehydrogenase
From Lehninger Principles of Biochemistry
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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Step 7 Hydration of Fumarate to Malate
Fumarase

• Hydration across the double bond
• trans-addition of the elements of water across
  the double bond
• the actual mechanism is not known for certain

From Lehninger Principles of Biochemistry
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Conversion of Fumarate to L-Malate goes through a
carbanion transition state
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Fumarase is highly stereospecific
trans configuration
cis configuration
fumarase
From Lehninger Principles of Biochemistry
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The Citric Acid Cycle has Eight Steps
From Lehninger Principles of Biochemistry
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Step 8 Oxidation of Malate to Oxaloacetate
Malate Dehydrogenase

• An NAD-dependent oxidation
• The carbon that gets oxidized is the one that
  received the -OH in the previous reaction
• This reaction is energetically expensive
• ?Go' 30 kJ/mol

From Lehninger Principles of Biochemistry
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Each turn of the citric acid cycle produces 3
NADH, 1 FADH, 1 GTP (or ATP), and 2 CO2
TCA Cycle
From Lehninger Principles of Biochemistry
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From Lehninger Principles of Biochemistry
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Citrate A Symmetrical Molecule That Reacts
Asymmetrically
From Lehninger Principles of Biochemistry
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From Lehninger Principles of Biochemistry
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Intermediates for Biosynthesis The TCA cycle
provides several of these
? -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
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Citric acid cycle components are important
biosynthetic intermediates
Amphibolic pathway, i.e., serves in both
catabolic anabolic processes
From Lehninger Principles of Biochemistry
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Anaplerotic reactions replenish citric acid cycle
intermediates
pyruvate
TCA Cycle
From Lehninger Principles of Biochemistry
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Under physiological conditions except for 1,3 4
all reaction have DG values closer to zero
Standard free energy changes for TCA cycle
???DG'? -32.2 kJ/mol
???DG'? 29.7 kJ/mol
???DG'? 13.3 kJ/mol
???DG'? -3.8 kJ/mol
???DG'? -20.9 kJ/mol
???DG'? 0 kJ/mol
???DG'? -33.5 kJ/mol
???DG'? -2.9 kJ/mol
From Lehninger Principles of Biochemistry
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Regulation of Citric Acid Cycle
The citric acid cycle is regulated at its three
exergonic steps
From Lehninger Principles of Biochemistry
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