Aconitase
| 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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| Structure of aconitase.[2] | |||||||||
| Identifiers | |||||||||
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| Symbol | Aconitase | ||||||||
| Pfam | PF00330 | ||||||||
| InterPro | IPR001030 | ||||||||
| PROSITE | PDOC00423 | ||||||||
| SCOP | 1aco | ||||||||
| SUPERFAMILY | 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 |
[edit] Function
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]
[edit] Enzyme 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.[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]
[edit] Enzyme Mechanism
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]
[edit] Disease Relevance
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]
[edit] Family members
Aconitases are expressed in bacteria to humans. Humans express the following two aconitase isozymes:
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[edit] Interactive pathway map
Click on genes, proteins and metabolites below to link to respective articles. [22]
[edit] References
- ^ 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.
- ^ 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.
- ^ Beinert, H; Kennedy, MC (1993). "Aconitase, a two-faced protein: Enzyme and iron regulatory factor". The FASEB journal 7 (15): 1442–9. PMID 8262329.
- ^ Flint, Dennis H.; Allen, Ronda M. (1996). "Iron−Sulfur Proteins with Nonredox Functions". Chemical Reviews 96 (7): 2315–34. doi:10.1021/cr950041r.
- ^ 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.
- ^ 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 9780121822521.
- ^ 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.
- ^ 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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=287193.
- ^ 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.
- ^ a b c d e f g h i Takusagawa F. "Chapter 16: Citric Acid Cycle". Takusagawa’s Note. The University of Kansas. http://web.ku.edu/~crystal/taksnotes/Biol_638/notes/chp_16.pdf. Retrieved 2011-07-10.
- ^ 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. http://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf.
- ^ 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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2144235.
- ^ 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.
- ^ 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.
- ^ "Aconitase family". The Prosthetic groups and Metal Ions in Protein Active Sites Database Version 2.0. The University of Leeds. 1999-02-02. http://metallo.scripps.edu/PROMISE/ACONITASE.html. Retrieved 2011-07-10.
- ^ a b c d Orphanet, "Aconitase deficiency," April 2008, http://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=43115
- ^ 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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=288463.
- ^ 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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2885827.
- ^ 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.
- ^ 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.
- ^ "Alloxan Diabetes - Medical Definition," Stedman's Medical Dictionary, 2006 Lippincott Williams & Wilkins, http://www.medilexicon.com/medicaldictionary.php?t=24313
- ^ The interactive pathway map can be edited at WikiPathways: "TCA_Cycle_WP78". http://www.wikipathways.org/index.php/Pathway:WP78.
[edit] Further reading
- 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.
[edit] External links
- MeSH Aconitase
- Proteopedia Aconitase - the Aconitase structure in interactive 3D
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