Aconitase

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aconitate hydratase
7ACN.jpg
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
Aconitase family
(aconitate hydratase)
PDB 1aco EBI.jpg
Structure of aconitase.[2]
Identifiers
Symbol Aconitase
Pfam PF00330
InterPro IPR001030
PROSITE PDOC00423
SCOP 1aco
SUPERFAMILY 1aco

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]

Function[edit]

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]

Enzyme Structure[edit]

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]

Enzyme Mechanism[edit]

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]

Disease Relevance[edit]

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]

Family members[edit]

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

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

Interactive pathway map[edit]

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

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TCACycle_WP78 go to article go to article go to article go to article go to HMDB go to article go to article go to article Go to article go to article go to article go to article go to article go to article Go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to HMDB go to article go to article go to HMDB go to article go to article go to HMDB go to article go to article go to HMDB go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to WikiPathways go to article go to article go to article go to article
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TCA Cycle edit
  1. ^ The interactive pathway map can be edited at WikiPathways: "TCACycle_WP78". 

References[edit]

  1. ^ PDB 7ACN; 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. doi:10.1021/bi00125a014. PMID 1547214. 
  2. ^ PDB 1ACO; 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. doi:10.1006/jmbi.1994.1246. PMID 8151704. 
  3. ^ Beinert, H; Kennedy, MC (1993). "Aconitase, a two-faced protein: Enzyme and iron regulatory factor". The FASEB Journal 7 (15): 1442–9. PMID 8262329. 
  4. ^ Flint, Dennis H.; Allen, Ronda M. (1996). "Iron−Sulfur Proteins with Nonredox Functions". Chemical Reviews 96 (7): 2315–34. doi:10.1021/cr950041r. 
  5. ^ Beinert, Helmut; Kennedy, Mary Claire; Stout, C. David (1996). "Aconitase as Iron−Sulfur Protein, Enzyme, and Iron-Regulatory Protein". Chemical Reviews 96 (7): 2335–74. doi:10.1021/cr950040z. PMID 11848830. 
  6. ^ Gardner, Paul R. (2002). "Aconitase: Sensitive target and measure of superoxide". Superoxide Dismutase. Methods in Enzymology 349. pp. 9–23. doi:10.1016/S0076-6879(02)49317-2. ISBN 978-0-12-182252-1. 
  7. ^ a b c d Robbins AH, Stout CD (1989). "The structure of aconitase". Proteins 5 (4): 289–312. doi:10.1002/prot.340050406. PMID 2798408. 
  8. ^ a b c Robbins AH, Stout CD (May 1989). "Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal". Proc. Natl. Acad. Sci. U.S.A. 86 (10): 3639–43. doi:10.1073/pnas.86.10.3639. PMC 287193. PMID 2726740. 
  9. ^ Lauble H, Kennedy MC, Beinert H, Stout CD (March 1992). "Crystal structures of aconitase with isocitrate and nitroisocitrate bound". Biochemistry 31 (10): 2735–48. doi:10.1021/bi00125a014. PMID 1547214. 
  10. ^ a b c d e f g h i Takusagawa F. "Chapter 16: Citric Acid Cycle". Takusagawa’s Note. The University of Kansas. Retrieved 2011-07-10. 
  11. ^ a b c Beinert H, Kennedy MC, Stout CD (November 1996). "Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein". Chem. Rev. 96 (7): 2335–2374. doi:10.1021/cr950040z. PMID 11848830. 
  12. ^ a b PDB 1C96; 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. doi:10.1110/ps.8.12.2655. PMC 2144235. PMID 10631981. 
  13. ^ Han D, Canali R, Garcia J, Aguilera R, Gallaher TK, Cadenas E (September 2005). "Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione". Biochemistry 44 (36): 11986–96. doi:10.1021/bi0509393. PMID 16142896. 
  14. ^ a b c Lauble H, Stout CD (May 1995). "Steric and conformational features of the aconitase mechanism". Proteins 22 (1): 1–11. doi:10.1002/prot.340220102. PMID 7675781. 
  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. 
  16. ^ a b c d Orphanet, "Aconitase deficiency," April 2008, http://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=43115
  17. ^ Hall, R E; Henriksson, K G; Lewis, S F; Haller, R G; Kennaway, N G (1993). "Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins". Journal of Clinical Investigation 92 (6): 2660–6. doi:10.1172/JCI116882. PMC 288463. PMID 8254022. 
  18. ^ a b c Ye, Hong; Rouault, Tracey A. (2010). "Human Iron−Sulfur Cluster Assembly, Cellular Iron Homeostasis, and Disease". Biochemistry 49 (24): 4945–56. doi:10.1021/bi1004798. PMC 2885827. PMID 20481466. 
  19. ^ a b c d e Boquist, L.; Ericsson, I.; Lorentzon, R.; Nelson, L. (1985). "Alterations in mitochondrial aconitase activity and respiration, and in concentration of citrate in some organs of mice with experimental or genetic diabetes". FEBS Letters 183 (1): 173–6. doi:10.1016/0014-5793(85)80979-0. PMID 3884379. 
  20. ^ a b c d e Lin, G.; Brownsey, R. W.; MacLeod, K. M. (2009). "Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart". Cellular and Molecular Life Sciences 66 (5): 919–32. doi:10.1007/s00018-009-8696-3. PMID 19153662. 
  21. ^ "Alloxan Diabetes - Medical Definition," Stedman's Medical Dictionary, 2006 Lippincott Williams & Wilkins, http://www.medilexicon.com/medicaldictionary.php?t=24313

Further reading[edit]

  • Frishman, Dmitrij; Hentze, Matthias W. (1996). "Conservation of Aconitase Residues Revealed by Multiple Sequence Analysis. Implications for Structure/Function Relationships". European Journal of Biochemistry 239 (1): 197–200. doi:10.1111/j.1432-1033.1996.0197u.x. PMID 8706708. 

External links[edit]