| 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] | |||||||||
| Identifiers | |||||||||
| EC no. | 4.2.1.3 | ||||||||
| CAS no. | 9024-25-3 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| Gene Ontology | AmiGO /QuickGO | ||||||||
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| Aconitase family (aconitate hydratase) | |||||||||
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 Structure of aconitase.[2] | |||||||||
| Identifiers | |||||||||
| Symbol | Aconitase | ||||||||
| Pfam | PF00330 | ||||||||
| InterPro | IPR001030 | ||||||||
| PROSITE | PDOC00423 | ||||||||
| SCOP2 | 1aco /SCOPe /SUPFAM | ||||||||
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Aconitase (aconitate hydratase;EC4.2.1.3) is an enzyme that catalyses thestereo-specificisomerization ofcitrate toisocitrate viacis-aconitate in thetricarboxylic acid cycle, a non-redox-active process.[3][4][5]
Aconitase 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 theN-terminus, only the first three of these domains are involved in close interactions with the [3Fe-4S] cluster, but theactive site consists of residues from all four domains, including the largerC-terminal domain.[6] The Fe-S cluster and aSO2−
4 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 ofiron-sulfur proteins that function as electron carriers, theiron-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. Threecysteine (Cys) residues have been shown to be ligands of the [Fe4S4] centre. In the active state, the labileiron ion of the [Fe4S4] cluster is not coordinated by Cys but by water molecules.
Theiron-responsive element-binding protein (IRE-BP) and3-isopropylmalate dehydratase (α-isopropylmalate isomerase;EC4.2.1.33), an enzyme catalysing the second step in the biosynthesis ofleucine, 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 inribosome binding and control themRNA 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 byserine, have no aconitase activity, but retain RNA-binding properties.
Aconitase is inhibited byfluoroacetate, 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.[9] The iron sulfur cluster is highly sensitive to oxidation bysuperoxide.[10]
Aconitase employs a dehydration-hydration mechanism.[11] The catalytic residues involved are His-101 and Ser-642.[11] 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, creating a double bond between C2 and C3, and forming the so-calledcis-aconitate intermediate (the twocarboxyl groups on the double bond arecis).[11][14] The carbon atom from which the hydrogen is removed is the one that came fromoxaloacetate in the previous step of the citric acid cycle, not the one that came fromacetyl CoA, even though these two carbons are equivalent except that one is "pro-R" and the other "pro-S" (seeProchirality).[15]: 393 At this point, the intermediate is rotated 180°.[11] This rotation is referred to as a "flip."[12] Because of this flip, the intermediate is said to move from a "citrate mode" to a "isocitrate mode."[16]
How exactly this flip occurs is debatable. One theory is that, in therate-limiting step of the mechanism, thecis-aconitate is released from the enzyme, then reattached in the isocitrate mode to complete the reaction.[16] This rate-limiting step ensures that the rightstereochemistry, specifically (2R,3S), is formed in the final product.[16][17] Another hypothesis is thatcis-aconitate stays bound to the enzyme while it flips from the citrate to the isocitrate mode.[11]
In either case, flippingcis-aconitate allows the dehydration and hydration steps to occur on opposite faces of the intermediate.[11] Aconitase catalyzestrans elimination/addition of water, and the flip guarantees that the correct stereochemistry is formed in the product.[11][12] 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 anucleophile to attack at C2, and the protonated serine is deprotonated by thecis-aconitate double bond to complete the hydration, producing isocitrate.[11]
Aconitases are expressed in bacteria to humans. Incitrus fruits, a reduction of the activity of the mitochondrial aconitases likely leads to the buildup of citric acid, which is then stored invacuoles.[18] 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.[18] Humans express the following two aconitaseisozymes:
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Click on genes, proteins and metabolites below to link to respective articles.[§ 1]
