View source for Aconitase

{{Distinguish|Aconitine}}
{{enzyme
| Name = aconitate hydratase
| EC_number = 4.2.1.3
| CAS_number = 9024-25-3
| IUBMB_EC_number = 4/2/1/3
| GO_code = 0003994
| image = 7ACN.jpg
| width = 
| caption = Illustration of  pig aconitase in complex with the [Fe<sub>4</sub>S<sub>4</sub>] cluster. The protein is colored by secondary structure, and iron atoms are blue and the sulfur red.<ref name="pmid1547214">{{PDB|7ACN}}; {{cite journal |pages=2735–48 |doi=10.1021/bi00125a014 |title=Crystal structures of aconitase with isocitrate and nitroisocitrate bound |year=1992 |last1=Lauble |first1=H. |last2=Kennedy |first2=M. C. |last3=Beinert |first3=H. |last4=Stout |first4=C. D. |journal=Biochemistry |volume=31 |issue=10 |pmid=1547214}}</ref>
}}
{{Infobox protein family
| Symbol = Aconitase
| Name = Aconitase family<br />(aconitate hydratase)
| image = PDB 1aco EBI.jpg
| width = 
| caption = Structure of aconitase.<ref name="pmid8151704">{{PDB|1ACO}}; {{cite journal |pages=437–51 |doi=10.1006/jmbi.1994.1246 |title=Crystal Structures of Aconitase with Trans-aconitate and Nitrocitrate Bound |year=1994 |last1=Lauble |first1=H |journal=Journal of Molecular Biology |volume=237 |issue=4 |pmid=8151704 |last2=Kennedy |first2=MC |last3=Beinert |first3=H |last4=Stout |first4=CD}}</ref>
| Pfam = PF00330
| InterPro = IPR001030
| SMART = 
| PROSITE = PDOC00423
| SCOP = 1aco
| TCDB = 
| OPM family = 
| OPM protein = 
}}
'''Aconitase''' (aconitate hydratase; {{EC number|4.2.1.3}}) is an enzyme that catalyses the [[stereochemistry|stereo-specific]] [[isomerization]] of [[citrate]] to [[isocitrate]] via ''cis''-[[aconitate]] in the [[tricarboxylic acid cycle]], a non-[[redox]]-active process.<ref name="pmid8262329">{{cite journal | vauthors = Beinert H, Kennedy MC | title = Aconitase, a two-faced protein: enzyme and iron regulatory factor | journal = FASEB Journal | volume = 7 | issue = 15 | pages = 1442–9 | date = Dec 1993 | pmid = 8262329 }}</ref><ref name="pmid11848829">{{cite journal |pages=2315–34 |doi=10.1021/cr950041r |title=Iron−Sulfur Proteins with Nonredox Functions |year=1996 |last1=Flint |first1=Dennis H. |last2=Allen |first2=Ronda M. |journal=Chemical Reviews |volume=96 |issue=7}}</ref><ref name="pmid11848830">{{cite journal | vauthors = Beinert H, Kennedy MC, Stout CD | title = Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein | journal = Chemical Reviews | volume = 96 | issue = 7 | pages = 2335–2374 | date = Nov 1996 | pmid = 11848830 | doi = 10.1021/cr950040z }}</ref>

<gallery>
 Image:Citrate wpmp.png|<center>[[Citric acid]]</center>
 Image:Cis-Aconitate wpmp.png|<center>[[Aconitic acid]]</center>
 Image:isocitric acid.svg|<center>[[Isocitric acid]]</center>
</gallery>

==Structure==
Aconitase, displayed in the structures in the right margin of this page, has two slightly different structures, depending on whether it is activated or inactivated.<ref name="inactive structure">{{cite journal | vauthors = Robbins AH, Stout CD | title = The structure of aconitase | journal = Proteins | volume = 5 | issue = 4 | pages = 289–312 | year = 1989 | pmid = 2798408 | doi = 10.1002/prot.340050406 }}</ref><ref name="active structure">{{cite journal | vauthors = Robbins AH, Stout CD | title = Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 86 | issue = 10 | pages = 3639–43 | date = May 1989 | pmid = 2726740 | pmc = 287193 | doi = 10.1073/pnas.86.10.3639 }}</ref> In the inactive form, its structure is divided into four domains.<ref name = "inactive structure"/> Counting from the [[N-terminus]], only the first three of these domains are involved in close interactions with the [3Fe-4S] cluster, but the [[active site]] consists of residues from all four domains, including the larger [[C-terminal]] domain.<ref name = "inactive structure"/> The Fe-S cluster and a SO<sub>4</sub><sup>2−</sup> anion also reside in the active site.<ref name = "inactive structure"/> When the enzyme is activated, it gains an additional iron atom, creating a [4Fe-4S] cluster.<ref name = "active structure" /><ref name="extra structure">{{cite journal | vauthors = Lauble H, Kennedy MC, Beinert H, Stout CD | title = Crystal structures of aconitase with isocitrate and nitroisocitrate bound | journal = Biochemistry | volume = 31 | issue = 10 | pages = 2735–48 | date = Mar 1992 | pmid = 1547214 | doi = 10.1021/bi00125a014 }}</ref> However, the structure of the rest of the enzyme is nearly unchanged; the conserved atoms between the two forms are in essentially the same positions, up to a difference of 0.1 angstroms.<ref name = "active structure"/>

== Function ==
In contrast with the majority of [[iron-sulfur protein]]s that function as electron carriers, the [[iron-sulfur cluster]] of aconitase reacts directly with an enzyme substrate. Aconitase has an active [Fe<sub>4</sub>S<sub>4</sub>]<sup>2+</sup> cluster, which may convert to an inactive [Fe<sub>3</sub>S<sub>4</sub>]<sup>+</sup> form. Three [[cysteine]] (Cys) residues have been shown to be ligands of the [Fe<sub>4</sub>S<sub>4</sub>] centre. In the active state, the labile [[iron]] ion of the [Fe<sub>4</sub>S<sub>4</sub>] cluster is not coordinated by Cys but by water molecules.

The [[iron-responsive element-binding protein]] (IRE-BP) and [[3-Isopropylmalate dehydratase|3-isopropylmalate dehydratase]] (α-isopropylmalate isomerase; {{EC number|4.2.1.33}}), an enzyme catalysing the second step in the biosynthesis of [[leucine]], are known aconitase homologues. Iron regulatory elements (IREs) constitute a family of 28-nucleotide, non-coding, stem-loop structures that regulate iron storage, [[heme]] synthesis and iron uptake. They also participate in [[ribosome]] binding and control the [[mRNA]] turnover (degradation). The specific regulator protein, the IRE-BP, binds to IREs in both 5' and 3' regions, but only to RNA in the apo form, without the Fe-S cluster. Expression of IRE-BP in cultured cells has revealed that the protein functions either as an active aconitase, when cells are iron-replete, or as an active RNA-binding protein, when cells are iron-depleted. Mutant IRE-BPs, in which any or all of the three Cys residues involved in Fe-S formation are replaced by [[serine]], have no aconitase activity, but retain RNA-binding properties.

Aconitase is inhibited by [[Fluoroacetic acid|fluoroacetate]], therefore fluoroacetate is poisonous. Fluoroacetate, in the citric acid cycle,can  innocently enter as fluorocitrate. However, aconitase cannot bind this substrate and thus the citric acid cycle is halted. The iron sulfur cluster is highly sensitive to oxidation by [[superoxide]].<ref name="pmid11912933">{{cite book |pages=9–23 |doi=10.1016/S0076-6879(02)49317-2 |chapter=Aconitase: Sensitive target and measure of superoxide |title=Superoxide Dismutase |series=Methods in Enzymology |year=2002 |last1=Gardner |first1=Paul R. |isbn=978-0-12-182252-1 |volume=349}}</ref>

===Mechanism===
[[File:Arrow Pushing Aconitase Final draft.tif|thumb|none|upright=2.5|Aconitase arrow-pushing mechanism <ref name = "Mechanism Source"/><ref name="Alchemy source"/>]]
[[File:Citrate Zoom Final.png|thumb|none|upright=1.5|Citrate and the Fe-S cluster in the active site of aconitase: dashed yellow lines show interactions between the substrate and nearby residues<ref name="pmid10631981">{{PDB|1C96}}; {{cite journal | vauthors = Lloyd SJ, Lauble H, Prasad GS, Stout CD | title = The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex | journal = Protein Sci. | volume = 8 | issue = 12 | pages = 2655–62 |date=December 1999 | pmid = 10631981 | pmc = 2144235 | doi = 10.1110/ps.8.12.2655 | url = }}</ref>]]

Aconitase employs a dehydration-hydration mechanism.<ref name = "Mechanism Source" /> The catalytic residues involved are His-101 and Ser-642.<ref name="Mechanism Source">{{cite web|url=http://web.ku.edu/~crystal/taksnotes/Biol_638/notes/chp_16.pdf |title=Chapter 16: Citric Acid Cycle |author=Takusagawa F |authorlink= |coauthors= |date= |format= |work=Takusagawa’s Note |publisher=The University of Kansas |accessdate=2011-07-10 |deadurl=yes |archiveurl=https://web.archive.org/web/20120324072437/http://web.ku.edu/~crystal/taksnotes/Biol_638/notes/chp_16.pdf |archivedate=2012-03-24 }}</ref> His-101 protonates the hydroxyl group on C3 of citrate, allowing it to leave as water, and Ser-642 concurrently abstracts the proton on C2, forming a double bond between C2 and C3, forming a ''cis''-aconitate intermediate.<ref name = "Mechanism Source" /><ref name = "ACS source">{{cite journal | vauthors = Han D, Canali R, Garcia J, Aguilera R, Gallaher TK, Cadenas E | title = Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione | journal = Biochemistry | volume = 44 | issue = 36 | pages = 11986–96 | date = Sep 2005 | pmid = 16142896 | doi = 10.1021/bi0509393 }}</ref>  At this point, the intermediate is rotated 180°.<ref name = "Mechanism Source" /> This rotation is referred to as a "flip."<ref name="Alchemy source">{{cite journal | vauthors = Beinert H, Kennedy MC, Stout CD | title = Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein | journal = Chemical Reviews | volume = 96 | issue = 7 | pages = 2335–2374 | date = Nov 1996 | pmid = 11848830 | doi = 10.1021/cr950040z | url = http://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf | access-date = 2011-05-16 | archive-url = https://web.archive.org/web/20110811075307/http://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf | archive-date = 2011-08-11 | dead-url = yes | df =  }}</ref> Because of this flip, the intermediate is said to move from a "citrate mode" to a "isocitrate mode."<ref name = "Different modes">{{cite journal | vauthors = Lauble H, Stout CD | title = Steric and conformational features of the aconitase mechanism | journal = Proteins | volume = 22 | issue = 1 | pages = 1–11 | date = May 1995 | pmid = 7675781 | doi = 10.1002/prot.340220102 }}</ref>

How exactly this flip occurs is debatable. One theory is that, in the [[rate-limiting step]] of the mechanism, the ''cis''-aconitate is released from the enzyme, then reattached in the isocitrate mode to complete the reaction.<ref name= "Different modes" /> This rate-liming step ensures that the right [[stereochemistry]], specifically (2R,3S), is formed in the final product.<ref name = "Different modes"/><ref name="Exact sterochem">{{cite web | url = http://metallo.scripps.edu/PROMISE/ACONITASE.html | title = Aconitase family | author =  | date = 1999-02-02 | work = The Prosthetic groups and Metal Ions in Protein Active Sites Database Version 2.0 | publisher = The University of Leeds | accessdate = 2011-07-10 | archiveurl = https://web.archive.org/web/20110608223412/http://metallo.scripps.edu/PROMISE/ACONITASE.html | archivedate = 2011-06-08 | deadurl = yes | df =  }}</ref> Another hypothesis is that ''cis''-aconitate stays bound to the enzyme while it flips from the citrate to the isocitrate mode.<ref name="Mechanism Source"/>

In either case, flipping ''cis''-aconitate allows the dehydration and hydration steps to occur on opposite faces of the intermediate.<ref name = "Mechanism Source" /> Aconitase catalyzes ''trans'' elimination/addition of water, and the flip guarantees that the correct stereochemistry is formed in the product.<ref name = "Mechanism Source" /><ref name = "Alchemy source" /> To complete the reaction, the serine and histidine residues reverse their original catalytic actions: the histidine, now basic, abstracts a proton from water, priming it as a [[nucleophile]] to attack at C2, and the protonated serine is deprotonated by the ''cis''-aconitate double bond to complete the hydration, producing isocitrate.<ref name = "Mechanism Source" />

[[File:Isocitrate Zoom Final.png|thumb|none|upright=1.5|Isocitrate and the Fe-S cluster in the active site of aconitase<ref name="pmid10631981"/>{{PDB|1C97}}; ]]

== Family members ==
Aconitases are expressed in bacteria to humans.  Humans express the following two aconitase [[isozyme]]s:

{|
| {{infobox protein
| Name = [[ACO1|aconitase 1, soluble]]
| caption = 
| image = 
| width = 
| HGNCid = 117
| Symbol = [[ACO1]]
| AltSymbols = IREB1
| EntrezGene = 48
| OMIM = 100880
| RefSeq = NM_002197
| UniProt = P21399
| PDB = 
| ECnumber = 4.2.1.3
| Chromosome = 9
| Arm = p
| Band = 21.1
| LocusSupplementaryData = 
}}
| {{infobox protein
| Name = [[ACO2|aconitase 2, mitochondrial]]
| caption = 
| image = 
| width = 
| HGNCid = 118
| Symbol = [[ACO2]]
| AltSymbols = ACONM
| EntrezGene = 50
| OMIM = 100850
| RefSeq = NM_001098
| UniProt = Q99798
| PDB = 
| ECnumber = 4.2.1.3
| Chromosome = 22
| Arm = q
| Band = 13.2
| LocusSupplementaryData = 
}}
|}

== Interactive pathway map ==
{{TCACycle WP78|highlight=Aconitase}}

== References ==
{{reflist|2}}

== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Frishman D, Hentze MW | title = Conservation of aconitase residues revealed by multiple sequence analysis. Implications for structure/function relationships | journal = European Journal of Biochemistry / FEBS | volume = 239 | issue = 1 | pages = 197–200 | date = Jul 1996 | pmid = 8706708 | doi = 10.1111/j.1432-1033.1996.0197u.x }}
{{refend}}

== External links ==
* {{MeshName|Aconitase}}
* {{Proteopedia|Aconitase}} - the Aconitase structure in interactive 3D

{{Citric acid cycle enzymes}}
{{Mitochondrial enzymes}}
{{Isomerase}}
{{Enzymes}}
{{Portal bar|Molecular and Cellular Biology|border=no}}

[[Category:EC 4.2.1]]
[[Category:Iron-sulfur proteins]]
[[Category:Moonlighting proteins]]