CHUAH CHA

1
Aconitase

• CHUAH CHA
• LINHDA NGUYEN
• HUY NGUYEN
• JI SUN YOON
• ONYEMACHI EGBUTA

2
Aconitase a.k.a. Citrate(Isocitrate)hydrolase
COO-
COO-
COO-
OH
COO-
OH
COO-
OH
COO-
COO-
COO-
COO-
Citrate
cis-Aconitate
Isocitrate
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Discovery

• Discovered in 1937 by C. Martius
• First discovered in mammals
• Aconitase is inactive when isolated
• Incubating with iron under reducing
  conditions restores activity
• Iron in aconitase is present as an iron-
  sulfur cluster
• Aconitase does NOT function in electron
  transfer

PDB 7acn
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holo form
apo form
PDB 2b3y
PDB 2ipy
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Classification

• mAcn mitochondrial aconitases
• Ref Nanda, S. K., et al. J Virol. 2001
• 75(7)3352-62.
• 2. cAcn cytosolic aconitases
• IRP1 iron regulatory protein 1
• IRP2 iron regulatory protein 2
• Ref Rouault, T. A. J Biol Chem.
  2006
• 2(8))406-14.
• AcnA bacterial aconitases A
• 3. AcnB bacterial aconitases B

With the exception of IRP2, these demonstrate
bifunctional role of aconitase
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Stereochemistry of the Reaction
Ref Lauble, H., et al. Proc Natl Acad Sci.
1996 9313699-703.
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PDB 7acn
PDB 1c96
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Structure of the Active Site

• Monomeric protein with an Fe-S cluster
• 754 amino acid residues
• MW 84 kDa

Aconitase
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Active Site
3Fe-4S
4Fe-4S2
Active
Inactive

• Active iron is lost as Fe (II) under oxidative
  stress or Fe starvation.
  
  
  
  
• Inactive form is highly unstable and will
  disassemble.
• The apo-form of cytosolic aconitase binds to RNA
  and acts as an iron regulatory protein.
• In the mitochondria, disassembly simply results
  in inactivity.

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Iron-Sulfur centre in aconitase
The active site in aconitate can accommodate
citrate, isocitrate, and the cis-aconitase in
either the citrate or isocitrate mode.
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Site of Catalysis
4 coordination sphere
6 coordination sphere
Substrate free state
Substrate bound

• Spectroscopic studies give precise Information of
  the interaction of substrate and cluster, detail
  binding of substrate to protein and the catalytic
  mechanism
• 3D structure determine from X-ray crystallography
  reveals the base involved in the dehydration
  reaction (His 101)

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Isomerization reaction catalysed by aconitase
Isocitrate

• Significance in TCA
• Aconitase is an important enzyme widely
  distributed in nature that catalyses the specific
  isomerization of citrate to isocitrate via
  consecutive dehydration-hydration reaction as
  shown below
• A non-redox reaction
• Arrange citrate to a more easily oxidized isomer

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Reaction Mechanism
1800 rotation
a
a
ß
ß
ß
a
H
H
His167
His101
His147

• Arg 580 anchors to ?-carboxyl of all substrate
• The Fe functions as a lewis acid in the
  elimination of hydroxyl from substrate
• Mutations in Ser642 to alanine and Asp165 to
  serine leads to a dramatic decrease in Vmax of
  over five orders of magnitude.
• Elimination of the substrate hydroxyl is
  facilitated through protonation by His 101 and by
  hydrogen bonding of Asp165 to both the hydroxyl
  of substrate and the water in Fe
• Serine is the catalytic base

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Citrate is a key molecule for many energy
metabolisms inside a cell
Tong and Rouault, Biometals (2007) 20549564
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Citrate Metabolism is closely related to the iron
metabolism, which essentially involves IRP1 and
IRP2.
Tong and Rouault, Biometals (2007) 20549564
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The Iron-Sulfur Cluster Switch
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The Iron-Sulfur Cluster Switch
IRP1 is the apo-form of aconitase, lacking
4Fe-4Scluster, which is responsible for the
catalysis at the active site. The interconversion
chemistry between IRP1 and aconitase is closely
related to iron level in cell. However, the
mechanism of interconversion between IRP1 and
c-aconitase is not known.
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IRP and IRE
Iron Regulatory Proteins Bind to IRE(iron
responsive element) of an mRNA and which is
responsible for repression either translation or
degradation. Iron Responsive Element Presides
in 3 or 5UTR region of mRNAs encoding proteins
for iron metabolism.
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Regulation of Ferritin Expression
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Regulation of Transferrin Receptor Expression
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IRPIRE Binding ferritin H IRE (bullfrog) bound
to IRP1
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IRPIRE Binding What the Structure Tells Us

• Two-point contact with the IRE one centered on
  the extra-helical C8 of the stem, and the other
  on the apical loop.
• The IRE binding sites generally coincide with the
  FeS cluster-binding region in aconitase
  utilizing many of the same amino acids, which
  explains the mutually exclusive nature of the two
  functionalities.

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Proposed Mechanism
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Oxidative Damage

• Hypothesis
• the characteristic changes of aging are a
  consequence of the accumulation of random
  oxidative damage to cellular molecules produced
  under normal physiological conditions.

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Identification of housefly mitochondrial
aconitase as a target of oxidative damage
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Mitochondrial Aconitase Activity
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Survivorship Curve
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Results

• Oxidative damage to protein accumulates
  selectively during aging of the fly
• Inactivation of aconitase results in shortened
  life-span

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Conclusion

• Mitochondrial aconitase is a monomeric protein
  that catalyzes the isomerization of citrate
  to isocitrate.
• The active site contains an Fe-S cluster and is
  inactivated by oxidative stress or Fe-poor
  conditions.
• When Fe-S cluster is disassembled in the active
  site of the c-aconitase, the enzyme is
  converted to IRP1 which is involved in regulating
  the expression of proteins involved in iron
  metabolism.
• However, removal of enzymatic Fe in m-aconitase
  will lead to enzyme inactivation, shutting
  down the TCA cycle and may result in death of
  organism.