| aconitate hydratase | |||||||
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| 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 number | 4.2.1.3 | ||||||
| CAS number | 9024-25-3 | ||||||
| Databases | |||||||
| IntEnz | IntEnz view | ||||||
| BRENDA | BRENDA entry | ||||||
| ExPASy | NiceZyme view | ||||||
| KEGG | KEGG entry | ||||||
| MetaCyc | metabolic pathway | ||||||
| PRIAM | profile | ||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||
| Gene Ontology | AmiGO / EGO | ||||||
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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 | ||||||||
| SCOP | 1aco | ||||||||
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Aconitase (aconitate hydratase; EC 4.2.1.3) 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]
Contents |
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 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 fluoroacetate, therefore fluoroacetate is poisonous. The iron sulfur cluster is highly sensitive to oxidation by superoxide.[6]
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.[7][8] In the inactive form, its structure is divided into four domains.[7] 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.[7] The Fe-S cluster and a SO42- anion also reside in the active site.[7] When the enzyme is activated, it gains an additional iron atom, creating a [4Fe-4S] cluster.[8][9] 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.[8]
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]
A serious ailment associated with aconitase is known as aconitase deficiency.[16] 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.[16] The main symptoms are myopathy and exercise intolerance; physical strain is lethal for some patients because it can lead to circulatory shock.[16][17] There are no known treatments for aconitase deficiency.[16]
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.[18] 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.[18] This deviance from homeostasis causes FRDA, a neurodegenerative disease for which no effective treatments have been found.[18]
Finally, aconitase is thought to be associated with diabetes.[19][20] Although the exact connection is still being determined, multiple theories exist.[19][20] In a study of organs from mice with alloxan diabetes (experimentally induced diabetes[21]) and genetic diabetes, lower aconitase activity was found to decrease the rates of metabolic reactions involving citrate, pyruvate, and malate.[19] In addition, citrate concentration was observed to be unusually high.[19] Since these abnormal data were found in diabetic mice, the study concluded that low aconitase activity is likely correlated with genetic and alloxan diabetes.[19] 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.[20] That is, it converts isocitrate back to cis-aconitate more rapidly than usual, but the forward reaction proceeds at the usual rate.[20] This imbalance may contribute to disrupted metabolism in diabetics.[20]
Aconitases are expressed in bacteria to humans. Humans express the following two aconitase isozymes:
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Click on genes, proteins and metabolites below to link to respective articles.[22]
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