Title: CHUAH CHA
1Aconitase
- CHUAH CHA
- LINHDA NGUYEN
- HUY NGUYEN
- JI SUN YOON
- ONYEMACHI EGBUTA
2Aconitase a.k.a. Citrate(Isocitrate)hydrolase
COO-
COO-
COO-
OH
COO-
OH
COO-
OH
COO-
COO-
COO-
COO-
Citrate
cis-Aconitate
Isocitrate
3Discovery
- 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
4holo form
apo form
PDB 2b3y
PDB 2ipy
5Classification
- 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
6Stereochemistry of the Reaction
Ref Lauble, H., et al. Proc Natl Acad Sci.
1996 9313699-703.
7PDB 7acn
PDB 1c96
8 Structure of the Active Site
- Monomeric protein with an Fe-S cluster
- 754 amino acid residues
- MW 84 kDa
Aconitase
9Active 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.
10Iron-Sulfur centre in aconitase
The active site in aconitate can accommodate
citrate, isocitrate, and the cis-aconitase in
either the citrate or isocitrate mode.
11Site 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)
12Isomerization 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
13Reaction 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(No Transcript)
15Citrate is a key molecule for many energy
metabolisms inside a cell
Tong and Rouault, Biometals (2007) 20549564
16Citrate Metabolism is closely related to the iron
metabolism, which essentially involves IRP1 and
IRP2.
Tong and Rouault, Biometals (2007) 20549564
17The Iron-Sulfur Cluster Switch
18The 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.
19IRP 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(No Transcript)
21Regulation of Ferritin Expression
22Regulation of Transferrin Receptor Expression
23IRPIRE Binding ferritin H IRE (bullfrog) bound
to IRP1
24IRPIRE 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.
25Proposed Mechanism
26Oxidative 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.
27Identification of housefly mitochondrial
aconitase as a target of oxidative damage
28Mitochondrial Aconitase Activity
29Survivorship Curve
30Results
- Oxidative damage to protein accumulates
selectively during aging of the fly - Inactivation of aconitase results in shortened
life-span
31Conclusion
- 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.