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{{Short description|Class of enzymes}}
{{enzyme▼
{{cs1 config|name-list-style=vanc}}
| Name = aconitate hydratase▼
<!--{{Distinguish|Aconitine}}: why would anyone confuse the two?-->
| EC_number = 4.2.1.3▼
▲{{infobox enzyme
▲| Name = aconitate hydratase
▲| EC_number = 4.2.1.3
| CAS_number = 9024-25-3
| GO_code = 0003994
| IUBMB_EC_number = 4/2/1/3
| GO_codeimage = 00039947ACN.jpg
| imagewidth = 7ACN.jpg =
| 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> ▼
| 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
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| OPM protein =
}}
'''Aconitase''' (aconitate hydratase; {{EC numberEnzExplorer|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">{{vcite2cite 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 | doi = 10.1096/fasebj.7.15.8262329 | pmid = 8262329 | s2cid = 1107246 | doi-access = free }}</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 |pmid=11848829}}</ref><ref name="pmid11848830">{{vcite2cite 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:Threo-Ds-isocitrateisocitric wpmpacid.pngsvg|<{{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">{{vcite2cite 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 | s2cid = 36219029 }}</ref><ref name="active structure">{{vcite2cite 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 | bibcode = 1989PNAS...86.3639R | doi-access = free }}</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 {{chem|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">{{vcite2cite 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 ==
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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, is converted to fluorocitrate by citrate synthase. Fluorocitrate competitively inhibits aconitase halting the citric acid cycle.<ref name="PMID 13208639">{{cite journal | vauthors = Morrison JF, Peters RA | title = Biochemistry of fluoroacetate poisoning: the effect of fluorocitrate on purified aconitase | journal = Biochem. J. | volume = 58| issue = 3| pages = 473–9|date=November 1954| doi = 10.1042/bj0580473 | pmid = 13208639| pmc = 1269923 }}</ref> 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|pmid=11912933 }}</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}}; {{vcite2cite 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 = https://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 access-date= 2011-07-10 |url-status=dead |archive-url=https://web.archive.org/web/20120324072437/https://web.ku.edu/~crystal/taksnotes/Biol_638/notes/chp_16.pdf |archive-date=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, formingcreating a double bond between C2 and C3, and forming athe so-called ''cis''-aconitate intermediate (the two [[carboxyl group]]s on the double bond are ''cis'').<ref name = "Mechanism Source" /><ref name = "ACS source">{{vcite2cite 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> The carbon atom from which the hydrogen is removed is the one that came from [[oxaloacetate]] in the previous step of the citric acid cycle, not the one that came from [[acetyl CoA]], even though these two carbons are equivalent except that one is "''pro''-R" and the other "''pro''-S" (see [[Prochirality]]).<ref name=Stryer1981>{{cite book|author=Lubert Stryer|author-link=Lubert Stryer|title=Biochemistry|date=1981 |edition=2nd|title-link=Biochemistry (Stryer)|pages=295–296}}</ref>{{rp|393}} At this point, the intermediate is rotated 180°.<ref name = "Mechanism Source" /> This rotation is referred to as a "flip."<ref name = "Alchemy source">{{vcite2cite 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 = https://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf | access-date = 2011-05-16 | archive-url = https://web.archive.org/web/20110811075307/https://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf | archive-date = 2011-08-11 | url-status = dead }}</ref> Because of this flip, the intermediate is said to move from a "citrate mode" to a "isocitrate mode."<ref name = "Different modes">{{vcite2cite 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 | s2cid = 43006515 }}</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-liminglimiting 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 = https://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 | accessdateaccess-date = 2011-07-10 | archiveurlarchive-url = httphttps:https://web.archive.org/web/20110608223412/https://metallo.scripps.edu/PROMISE/ACONITASE.html | archivedatearchive-date = 8 June 2011 <!-06-DASHBot-->08 | deadurlurl-status = nodead }}</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}}; {{vcite2 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 }}</ref>]]
 
==Clinical significance==
A serious ailment associated with aconitase is known as aconitase deficiency.<ref name = "Orphanet: Aconitase deficiency">Orphanet, "Aconitase deficiency," April 2008, https://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=43115</ref> It is caused by a mutation in the gene for iron-sulfur cluster scaffold protein ([[ISCU]]), which helps build the Fe-S cluster on which the activity of aconitase depends.<ref name = "Orphanet: Aconitase deficiency" /> The main symptoms are [[myopathy]] and [[exercise intolerance]]; physical strain is lethal for some patients because it can lead to [[circulatory shock]].<ref name = "Orphanet: Aconitase deficiency" /><ref name=pmid8254022>{{vcite2 journal | vauthors = Hall RE, Henriksson KG, Lewis SF, Haller RG, Kennaway NG | title = Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins | journal = The Journal of Clinical Investigation | volume = 92 | issue = 6 | pages = 2660–6 | date = Dec 1993 | pmid = 8254022 | pmc = 288463 | doi = 10.1172/JCI116882 }}</ref> There are no known treatments for aconitase deficiency.<ref name = "Orphanet: Aconitase deficiency" />
 
Another disease associated with aconitase is [[Friedreich's ataxia]] (FRDA), which is caused when the Fe-S proteins in aconitase and [[succinate dehydrogenase]] have decreased activity.<ref name=pmid20481466>{{vcite2 journal | vauthors = Ye H, Rouault TA | title = Human iron-sulfur cluster assembly, cellular iron homeostasis, and disease | journal = Biochemistry | volume = 49 | issue = 24 | pages = 4945–56 | date = Jun 2010 | pmid = 20481466 | pmc = 2885827 | doi = 10.1021/bi1004798 }}</ref> A proposed mechanism for this connection is that decreased Fe-S activity in aconitase and succinate dehydrogenase is correlated with excess iron concentration in the mitochondria and insufficient iron in the cytoplasm, disrupting [[iron homeostasis]].<ref name=pmid20481466/> This deviance from homeostasis causes FRDA, a [[neurodegenerative disease]] for which no effective treatments have been found.<ref name=pmid20481466/>
 
Finally, aconitase is thought to be associated with [[diabetes]].<ref name=pmid3884379/><ref name = "type 1 source"/> Although the exact connection is still being determined, multiple theories exist.<ref name=pmid3884379>{{vcite2 journal | vauthors = Boquist L, Ericsson I, Lorentzon R, Nelson L | title = Alterations in mitochondrial aconitase activity and respiration, and in concentration of citrate in some organs of mice with experimental or genetic diabetes | journal = FEBS Letters | volume = 183 | issue = 1 | pages = 173–6 | date = Apr 1985 | pmid = 3884379 | doi = 10.1016/0014-5793(85)80979-0 }}</ref><ref name= "type 1 source"/> In a study of organs from mice with alloxan diabetes (experimentally induced diabetes<ref name="alloxan def">"Alloxan Diabetes - Medical Definition," Stedman's Medical Dictionary, 2006 Lippincott Williams & Wilkins, https://www.medilexicon.com/medicaldictionary.php?t=24313</ref>) and genetic diabetes, lower aconitase activity was found to decrease the rates of metabolic reactions involving citrate, pyruvate, and malate.<ref name=pmid3884379/> In addition, citrate concentration was observed to be unusually high.<ref name=pmid3884379/> Since these abnormal data were found in diabetic mice, the study concluded that low aconitase activity is likely correlated with genetic and alloxan diabetes.<ref name=pmid3884379/> Another theory is that, in diabetic hearts, accelerated phosphorylation of heart aconitase by protein kinase C causes aconitase to speed up the final step of its reverse reaction relative to its forward reaction.<ref name = "type 1 source"/> That is, it converts isocitrate back to ''cis''-aconitate more rapidly than usual, but the forward reaction proceeds at the usual rate.<ref name = "type 1 source"/> This imbalance may contribute to disrupted metabolism in diabetics.<ref name = "type 1 source">{{vcite2 journal | vauthors = Lin G, Brownsey RW, MacLeod KM | title = Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart | journal = Cellular and Molecular Life Sciences | volume = 66 | issue = 5 | pages = 919–32 | date = Mar 2009 | pmid = 19153662 | doi = 10.1007/s00018-009-8696-3 }}</ref>
 
The mitochondrial form of aconitase, ACO2, is correlated with many diseases, as it is directly involved in the conversion of [[glucose]] into [[ATP]], or the central metabolic pathway. Decreased expression of ACO2 in gastric cancer cells has been associated with a poor prognosis;<ref>{{vcite2 journal | vauthors = Wang P, Mai C, Wei YL, Zhao JJ, Hu YM, Zeng ZL, Yang J, Lu WH, Xu RH, Huang P | title = Decreased expression of the mitochondrial metabolic enzyme aconitase (ACO2) is associated with poor prognosis in gastric cancer | journal = Medical Oncology | volume = 30 | issue = 2 | pages = 552 | date = Jun 2013 | pmid = 23550275 }}</ref> this effect has also been seen in prostate cancer cells.<ref>{{vcite2 journal | vauthors = Juang HH | title = Modulation of mitochondrial aconitase on the bioenergy of human prostate carcinoma cells | journal = Molecular Genetics and Metabolism | volume = 81 | issue = 3 | pages = 244–52 | date = Mar 2004 | pmid = 14972331 | doi = 10.1016/j.ymgme.2003.12.009 }}</ref><ref>{{vcite2 journal | vauthors = Tsui KH, Feng TH, Lin YF, Chang PL, Juang HH | title = p53 downregulates the gene expression of mitochondrial aconitase in human prostate carcinoma cells | journal = The Prostate | volume = 71 | issue = 1 | pages = 62–70 | date = Jan 2011 | pmid = 20607720 | doi = 10.1002/pros.21222 }}</ref> A few treatments have been identified in vitro to induce greater ACO2 expression, including exposing the cells to hypoxia and the element [[manganese]].<ref>{{vcite2 journal | vauthors = Tsui KH, Chung LC, Wang SW, Feng TH, Chang PL, Juang HH | title = Hypoxia upregulates the gene expression of mitochondrial aconitase in prostate carcinoma cells | journal = Journal of Molecular Endocrinology | volume = 51 | issue = 1 | pages = 131–41 | date = 2013 | pmid = 23709747 | doi = 10.1530/JME-13-0090 }}</ref><ref>{{vcite2 journal | vauthors = Tsui KH, Chang PL, Juang HH | title = Manganese antagonizes iron blocking mitochondrial aconitase expression in human prostate carcinoma cells | journal = Asian Journal of Andrology | volume = 8 | issue = 3 | pages = 307–15 | date = May 2006 | pmid = 16625280 | doi = 10.1111/j.1745-7262.2006.00139.x }}</ref>
 
== Family members ==
Aconitases are expressed in bacteria to humans. In [[citrus fruits]], a reduction of the activity of the mitochondrial aconitases likely leads to the buildup of citric acid, which is then stored in [[vacuole]]s.<ref name="Degu">{{cite journal |last1=Degu |first1=Asfaw |last2=Hatew |first2=Bayissa |last3=Nunes-Nesi |first3=Adriano |last4=Shlizerman |first4=Ludmila |last5=Zur |first5=Naftali |last6=Katz |first6=Ehud |last7=Fernie |first7=Alisdair R. |last8=Blumwald |first8=Eduardo |last9=Sadka |first9=Avi |title=Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis |journal=Planta |date=September 2011 |volume=234 |issue=3 |pages=501–513 |doi=10.1007/s00425-011-1411-2}}</ref> As the fruit matures, citric acid is returned back to the cytosol where an increase in cytosolic aconitase activity reduces its levels in the fruit.<ref name="Degu"/> Humans express the following two aconitase [[isozyme]]s:
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
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}}
| {{infobox protein
| Name = [[ACO2|aconitase 2, mitochondrial]]
| caption =
| image =
| width =
| HGNCid = 118
| Symbol = [[ACO2]]
| AltSymbols = ACONM
| EntrezGene = 50
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== Further reading ==
{{refbegin}}
* {{vcite2cite 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 | doi-access = free }}
{{refend}}
 
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* {{Proteopedia|Aconitase}} - the Aconitase structure in interactive 3D
 
{{Carbon-oxygen lyases}}▼
{{Citric acid cycle enzymes}}
{{Mitochondrial enzymes}}
▲{{Carbon-oxygen lyases}}
{{Enzymes}}
{{Portal bar|Biology|border=no}}
 
[[Category:EC 4.2.1]]
[[Category:Iron-sulfurIron–sulfur proteins]]
[[Category:Moonlighting proteins]]
Retrieved from "https://en.wikipedia.org/wiki/Aconitase"