Aconitase: Difference between revisions
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'''Aconitase (ACO) | <StructureSection load='' size='350' side='right' scene='Aconitase/Cv/1' caption='Bovine aconitase showing FeS4 cluster complex with sulfate (PDB code [[1amj]])'> | ||
==Function== | |||
[[Aconitase]] (ACO, EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.3 4.2.1.3]) is an enzymatic domain that confers the ability to catalyse the equilibrium | |||
:citrate = aconitate + H<sub>2</sub>O = L-isocitrate | :citrate = aconitate + H<sub>2</sub>O = L-isocitrate | ||
This reaction is part of the citrate (TCA-, Krebs-)cycle. | This reaction is part of the citrate (TCA-, Krebs-)cycle. | ||
In most organisms, there is a cytosolic enzyme with an ACO domain (cAc), and in eukaryotes, a second copy of it was introduced with mitochondria (mAc). Plants developed even more copies in mitochondria. | |||
Aconitase contains a Fe4S4 cluster which converts to Fe3S4 when the enzyme is inactive. In humans, two types of ACO are expressed: the soluble '''ACO1''' and the mitochondrial '''ACO2'''. Two types of '''ACO X''' were characterized as '''mevalonate 5-phosphate dehydratase''' and '''cis-3-hydroxy-L-proline dehydrates'''. | |||
Aconitase from pig (PDB [[7acn]]) is a single polypeptide (M<sub>r</sub> 83kD) that catalyzes the reversible isomerization of citrate and isocitrate.<ref name="Zheng">PMID 1313811</ref> It is the second enzyme in the Citric acid cycle, which is a series of enzyme-catalysed chemical reactions that is crucial to aerobic cellular respiration and the production of ATP. See also:<br /> | |||
*[[Citric Acid Cycle]] | |||
*[[Krebs cycle step 2]] | |||
*[[Glyoxylate cycle]] | |||
==Structure== | |||
The <scene name='Anthony_Noles_Sandbox/Secondary_structure/1'>secondary structure</scene> consists of numerous alternating alpha helices and beta sheets (SCOP classification α/β alternating). The tertiary structure is somewhat bilobed with the active site in the middle, and, since there is only one subunit, there is no quaternary structure. Aconitase consists of four domains, three of which are tightly packed while the fourth is more flexible. <ref name="Frishman">PMID 8706708</ref> Aconitase contains a <scene name='Anthony_Noles_Sandbox/Fe-scluster/2'>4Fe-4S iron-sulfur cluster</scene>. This iron sulfur cluster does not participate in redox as most do, but holds the OH group of citrate to facilitate its elimination.<ref>PMID:16407072 </ref> It is at this 4Fe-4S site that catalysis occurs and citrate or <scene name='Anthony_Noles_Sandbox/Fe-scluster_bound_isocitrate/8'>isocitrate</scene> is bound. The rest of the <scene name='Anthony_Noles_Sandbox/Fe-scluster_w_active_site/5'>active site (manually rotate this scene to see the proximity of each residue to the 4Fe-4S cluster)</scene> is made up of residues Gln72, Asp100, His101, Asp165, Ser166, His167, His147, Glu262, Asn258, Cys358, Cys421, Cys424, Cys358, Cys421, Asn446, Arg447, Arg452, Asp568, Ser642, Ser643, Arg644, Arg580. <ref name="Beinert">Beinert, H., Kennedy, M. C., Stout, C.D. “Aconitase as Iron−Sulfur Protein, Enzyme, and Iron-Regulatory Protein.” Chem. Rev. 1996, 96, 2335−2373.</ref> | |||
{{Clear}} | |||
== Catalytic mechanism of mitochondrial ACO == | == Catalytic mechanism of mitochondrial ACO == | ||
Both mAc and cAc are quite similar in their ACO function. Studies, however, concentrated on <scene name='Aconitase/7acn-sf4/1'>the mitochondrial ACO</scene>. ACO is an excellent system for understanding the role of iron-sulfur-clusters in catalysis. The <scene name='Aconitase/7acn-sf4/2'>(4Fe-4S) cofactor is held in place</scene> by three sulfur atoms belonging to the cysteins-385, -448, and -451 <scene name='33/338089/7acn-morph/5'>which are bound to three of the four</scene> cluster iron atoms. On activation of the enzyme, <scene name='33/338089/7acn-morph/8'>a fourth iron atom is included in the cluster</scene> together with a water molecule.This Fe4 is free to bind one, two, or three partners, in this reaction always oxygen atoms belonging to other molecules.<ref>PMID:8151704</ref> | |||
<!--It is clear that, in order to synthesize L-isocitrate, stereoselective catalysis must occur.--> | |||
Substrate-free aconitase contains a [4Fe-4S]<sup>2+</sup> cluster with hydroxyl bound to one of the Fe. Upon binding of substrate the bound hydroxyl is protonated. A hydrogen bond from <scene name='Anthony_Noles_Sandbox/His101/3'>His101</scene> to the isocitrate hydroxyl is donated to form water. Alternatively, the proton could be donated by <scene name='Anthony_Noles_Sandbox/His167/3'>His167</scene> as this histidine is hydrogen bonded to a H<sub>2</sub>O molecule. His167 is also hydrogen bonded to the bound H<sub>2</sub>O in the [4Fe-4S] cluster. Both <scene name='Anthony_Noles_Sandbox/His_101_and_167/4'>His101 and His167</scene> are paired with carboxylates (<scene name='Anthony_Noles_Sandbox/Asp100_and_glu262/3'>Asp100 and Glu262</scene>, respectively) and are likely to be protonated. The conformational change associated with substrate binding reorients the cluster. <ref name="Beinert" /> The residue which removes a proton from citrate or isocitrate is <scene name='Anthony_Noles_Sandbox/Ser642/4'>Ser642</scene>. <ref name="Beinert" /> This causes the cis-Aconitate intermediate (seen below), which consists of a double bond, which is a direct result of the deprotonation. Then, there is a rehydration of the double bond of cis-aconitate to form isocitrate (if the original substrate was citrate). To better understand this, consider this process as stages, seen below. | |||
{{Clear}} | |||
====Stage 1: Dehydration==== | |||
First, dehydration of citrate causes a proton and OH group to be removed from only the 'lower arm'.<ref name="Voet">Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry Life at the Molecular Level. New York: John Wiley & Sons, 2008. p. 578-579. Print.</ref> This forms a cis-Aconitate intermediate. | |||
====Stage 2: Rehydration==== | |||
The second main stage of the reaction is the rehydration of the cis-Aconitate intermediate. This forms isocitrate. It is catalyzed in a stereospecific way such that only one isocitrate stereoisomer is formed. <ref name="Voet" /> | |||
Thus, the overall reaction that aconitase catalyzes is: Citrate ←→cis-Aconitate←→Isocitrate, as seen below: | |||
[[ Image:Aconitase.JPG]] | |||
A more stereochemical view at the reaction shows that the aconitate intermediate has to make a 180 degree flip in order to start the other half of the reaction: | |||
[[Image:Aconitase steps stereo.png|400px]] | |||
==Regulation== | |||
Aconitase can be inhibited or activated to increase or decrease the ability to catalyze the reaction of citrate to isocitrate. The activity of aconitase can be reduced when one Fe is lost from the cluster. This lowers the activity over 100-fold, but then can regain full activity by adding another Fe from solution. <ref name="Flint">Flint, DH., and Allen, RM. "Iron-sulfur protein with nonredox functions.” Chem. Rev. 1996, 96, 2315−2334.</ref> Aconitase is also strongly inhibited by nitro analogs <ref name="Flint" /> | |||
The Citric Acid Cycle works in such a way that the product of one reaction becomes the reactant of another, with different enzymes catalyzing each reaction. Aconitase is one such enzyme. Some of these enzymes are tightly regulated, either activated or inhibited, by the concentration of reactant, product, ATP or NADH, and thus are rate-determining. Aconitase is not one of the three rate-determining enzymes of the Citric Acid Cycle as its ΔG is not negative (ΔG°′≈5 kJ/mol and ΔG≈0 kJ/mol).<ref name="Voet" /> Aconitase functions close to equilibrium and the rate of citrate consumption depends on the activity of NAD<sup>+</sup>-dependent isocitrate dehydrogenase, which is one of the three rate-determining enyzmes. Isocitrate dehydrogenase uses the product of the reaction aconitase catalyzes. Both Citrate synthase and Isocitrate dehydogenase are inhibited by NADH concentration, but aconitase itself is not.<ref name="Voet" /> Since the rate of aconitase depends on the activity of NAD<sup>+</sup>-dependent isocitrate dehydrogenase, then citrate could build up on the reactant side, which would then inhibit the enzyme of the previous step, citrate synthase. An illustration of this is seen below, with the boxes representing the enzymes that are catalyzing each reaction. This is a common example of how the Citric Acid Cycle works in order to produce ATP without wasting resources. Similar inhibition/activation of enzymes occurs based on concentrations of ATP, NADH, Calcium, CoA, and others. | |||
[[Image:Regulation.JPG|left|450px|thumb]] | |||
{{Clear}} | |||
== Cytosolic aconitase and its other function == | == Cytosolic aconitase and its other function == | ||
A specialty of cAc is that in mammals it has developed a <scene name='33/338089/Cv/2'>second function</scene> as inhibitor of <scene name='33/338089/Cv/3'>those mRNA</scene> that carry an <scene name='33/338089/Cv/4'>iron-responsive element (IRE)</scene>. Therefore, the cytosolic cAc is named IREBP for IRE-binding protein when this function is talked about. Only one of the two functions is active, depending on whether <scene name='Aconitase/2b3x-cluster/1'>the (4Fe-4S) cofactor</scene> is present in the molecule: it's essential for <scene name='Aconitase/2b3x-total/1'>the ACO function</scene>. You can see, by <scene name='Aconitase/Morph/2'>looking at the morph</scene>, how much the enzyme structure differs between those two functions. | |||
Along with serving as a catalyst, aconitase is a member of the iron regulatory protien-1 (IRP-1) family. These enzymes have been found to play a role in regulatory RNA-binding proteins. This suggests a novel role for Fe-S clusters as post-translational regulatory switches.<ref name="Frishman" /> | |||
== 3D structures of Aconitase== | |||
[[Aconitase 3D structures]] | |||
</StructureSection> | |||
__NOTOC__ | |||
== Literature == | |||
* M. Claire Kennedy and Helmut Beinert: ''IX.4. Aconitase.'' in Ivano Bertini, Harry B. Gray, Edward I. Stiefel, Joan Selverstone Valentine (eds.): ''Biological Inorganic Chemistry: Structure and Reactivity.'' University Science Books, Herndon 2006. ISBN 1891389432 pp.209-- | |||
==Additional Resources== | |||
For additional information, see: [[Carbohydrate Metabolism]]; [[Krebs cycle step 2]]. | |||
<br /> | |||
== References == | |||
<references/> | |||
== External links == | |||
== | |||
*[http://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb89_1.html Molecule of the Month: Aconitase and Iron Regulatory Protein 1] | *[http://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb89_1.html Molecule of the Month: Aconitase and Iron Regulatory Protein 1] | ||
*[http://en.wikipedia.org/wiki/Aconitase Aconitase at Wikipedia] | *[http://en.wikipedia.org/wiki/Aconitase Aconitase at Wikipedia] | ||
[[Category:Topic Page]] | |||