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CHUAH CHA

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Discovered in 1937 by C. Martius. First discovered in mammals ... ferritin H IRE (bullfrog) bound to IRP1. Two-point contact with the IRE: ... – PowerPoint PPT presentation

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Title: 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
3
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
4
holo form
apo form
PDB 2b3y
PDB 2ipy
5
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
6
Stereochemistry of the Reaction
Ref Lauble, H., et al. Proc Natl Acad Sci.
1996 9313699-703.
7
PDB 7acn
PDB 1c96
8
Structure of the Active Site
  • Monomeric protein with an Fe-S cluster
  • 754 amino acid residues
  • MW 84 kDa

Aconitase
9
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.

10
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.
11
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)

12
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

13
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

14
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15
Citrate is a key molecule for many energy
metabolisms inside a cell
Tong and Rouault, Biometals (2007) 20549564
16
Citrate Metabolism is closely related to the iron
metabolism, which essentially involves IRP1 and
IRP2.
Tong and Rouault, Biometals (2007) 20549564
17
The Iron-Sulfur Cluster Switch
18
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.
19
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.
20
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21
Regulation of Ferritin Expression
22
Regulation of Transferrin Receptor Expression
23
IRPIRE Binding ferritin H IRE (bullfrog) bound
to IRP1
24
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.

25
Proposed Mechanism
26
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.

27
Identification of housefly mitochondrial
aconitase as a target of oxidative damage
28
Mitochondrial Aconitase Activity
29
Survivorship Curve
30
Results
  • Oxidative damage to protein accumulates
    selectively during aging of the fly
  • Inactivation of aconitase results in shortened
    life-span

31
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.
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