aconitate hydratase
Illustration of pig aconitase in complex with the [Fe4S4] cluster. The protein is colored by secondary structure, and iron atoms are blue and the sulfur red.[1]
EC number
CAS number 9024-25-3
IntEnz IntEnz view
ExPASy NiceZyme view
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Aconitase family
(aconitate hydratase)
Structure of aconitase.[2]
Symbol Aconitase
Pfam PF00330
InterPro IPR001030
SCOP 1aco

Aconitase (aconitate hydratase; EC is an enzyme that catalyses the stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process.[3][4][5]


  • Structure 1
  • Function 2
    • Mechanism 2.1
  • Family members 3
  • Interactive pathway map 4
  • References 5
  • Further reading 6
  • External links 7


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.[6][7] In the inactive form, its structure is divided into four domains.[6] 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.[6] The Fe-S cluster and a SO42− anion also reside in the active site.[6] When the enzyme is activated, it gains an additional iron atom, creating a [4Fe-4S] cluster.[7][8] 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.[7]


In contrast with the majority of iron-sulfur proteins that function as electron carriers, the iron-sulfur cluster of aconitase reacts directly with an enzyme substrate. Aconitase has an active [Fe4S4]2+ cluster, which may convert to an inactive [Fe3S4]+ form. Three cysteine (Cys) residues have been shown to be ligands of the [Fe4S4] centre. In the active state, the labile iron ion of the [Fe4S4] cluster is not coordinated by Cys but by water molecules.

The iron-responsive element-binding protein (IRE-BP) and 3-isopropylmalate dehydratase (α-isopropylmalate isomerase; EC, 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 fluoroacetate, therefore fluoroacetate is poisonous. The iron sulfur cluster is highly sensitive to oxidation by superoxide.[9]


Aconitase arrow-pushing mechanism [10][11]
Citrate and the Fe-S cluster in the active site of aconitase: dashed yellow lines show interactions between the substrate and nearby residues[12]

Aconitase employs a dehydration-hydration mechanism.[10] The catalytic residues involved are His-101 and Ser-642.[10] 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.[10][13] At this point, the intermediate is rotated 180°.[10] This rotation is referred to as a "flip."[11] Because of this flip, the intermediate is said to move from a "citrate mode" to a "isocitrate mode."[14]

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.[14] This rate-liming step ensures that the right stereochemistry, specifically (2R,3S), is formed in the final product.[14][15] Another hypothesis is that cis-aconitate stays bound to the enzyme while it flips from the citrate to the isocitrate mode.[10]

In either case, flipping cis-aconitate allows the dehydration and hydration steps to occur on opposite faces of the intermediate.[10] Aconitase catalyzes trans elimination/addition of water, and the flip guarantees that the correct stereochemistry is formed in the product.[10][11] 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.[10]

Isocitrate and the Fe-S cluster in the active site of aconitase[12]​;

Family members

Aconitases are expressed in bacteria to humans. Humans express the following two aconitase isozymes:

aconitase 1, soluble
Symbol ACO1
Alt. symbols IREB1
Entrez 48
HUGO 117
OMIM 100880
RefSeq NM_002197
UniProt P21399
Other data
EC number
Locus Chr. 9 p21.1
aconitase 2, mitochondrial
Symbol ACO2
Alt. symbols ACONM
Entrez 50
HUGO 118
OMIM 100850
RefSeq NM_001098
UniProt Q99798
Other data
EC number
Locus Chr. 22 q13.2

Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles. [§ 1]

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TCA Cycle edit
  1. ^ The interactive pathway map can be edited at WikiPathways: "TCACycle_WP78". 


  1. ^ ​; Lauble, H.; Kennedy, M. C.; Beinert, H.; Stout, C. D. (1992). "Crystal structures of aconitase with isocitrate and nitroisocitrate bound". Biochemistry 31 (10): 2735–48.  
  2. ^ ​; Lauble, H; Kennedy, MC; Beinert, H; Stout, CD (1994). "Crystal Structures of Aconitase with Trans-aconitate and Nitrocitrate Bound". Journal of Molecular Biology 237 (4): 437–51.  
  3. ^ Beinert H, Kennedy MC (Dec 1993). "Aconitase, a two-faced protein: enzyme and iron regulatory factor". FASEB Journal 7 (15): 1442–9.  
  4. ^ Flint, Dennis H.; Allen, Ronda M. (1996). "Iron−Sulfur Proteins with Nonredox Functions". Chemical Reviews 96 (7): 2315–34.  
  5. ^ Beinert H, Kennedy MC, Stout CD (Nov 1996). "Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein". Chemical Reviews 96 (7): 2335–2374.  
  6. ^ a b c d Robbins AH, Stout CD (1989). "The structure of aconitase". Proteins 5 (4): 289–312.  
  7. ^ a b c Robbins AH, Stout CD (May 1989). "Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal". Proceedings of the National Academy of Sciences of the United States of America 86 (10): 3639–43.  
  8. ^ Lauble H, Kennedy MC, Beinert H, Stout CD (Mar 1992). "Crystal structures of aconitase with isocitrate and nitroisocitrate bound". Biochemistry 31 (10): 2735–48.  
  9. ^ Gardner, Paul R. (2002). "Aconitase: Sensitive target and measure of superoxide". Superoxide Dismutase. Methods in Enzymology 349. pp. 9–23.  
  10. ^ a b c d e f g h i Takusagawa F. "Chapter 16: Citric Acid Cycle" (PDF). Takusagawa’s Note. The University of Kansas. Retrieved 2011-07-10. 
  11. ^ a b c Beinert H, Kennedy MC, Stout CD (Nov 1996). "Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein" (PDF). Chemical Reviews 96 (7): 2335–2374.  
  12. ^ a b ​; Lloyd SJ, Lauble H, Prasad GS, Stout CD (December 1999). "The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex". Protein Sci. 8 (12): 2655–62.  
  13. ^ Han D, Canali R, Garcia J, Aguilera R, Gallaher TK, Cadenas E (Sep 2005). "Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione". Biochemistry 44 (36): 11986–96.  
  14. ^ a b c Lauble H, Stout CD (May 1995). "Steric and conformational features of the aconitase mechanism". Proteins 22 (1): 1–11.  
  15. ^ "Aconitase family". The Prosthetic groups and Metal Ions in Protein Active Sites Database Version 2.0. The University of Leeds. 1999-02-02. Archived from the original on 8 June 2011. Retrieved 2011-07-10. 

Further reading

  • Frishman D, Hentze MW (Jul 1996). "Conservation of aconitase residues revealed by multiple sequence analysis. Implications for structure/function relationships". European Journal of Biochemistry / FEBS 239 (1): 197–200.  

External links