aconitase

Aconitase has two slightly different structures, depending on whether it is activated or inactivated.{{cite journal | vauthors = Robbins AH, Stout CD | title = The structure of aconitase | journal = Proteins | volume = 5 | issue = 4 | pages = 289–312 | year = 1989 | pmid = 2798408 | doi = 10.1002/prot.340050406 | s2cid = 36219029 }}{{cite journal | vauthors = Robbins AH, Stout CD | title = Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 86 | issue = 10 | pages = 3639–43 | date = May 1989 | pmid = 2726740 | pmc = 287193 | doi = 10.1073/pnas.86.10.3639 | bibcode = 1989PNAS...86.3639R | doi-access = free }} In the inactive form, its structure is divided into four domains. 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. The Fe-S cluster and a {{chem|SO|4|2−}} anion also reside in the active site. When the enzyme is activated, it gains an additional iron atom, creating a [4Fe-4S] cluster.{{cite journal | vauthors = Lauble H, Kennedy MC, Beinert H, Stout CD | title = Crystal structures of aconitase with isocitrate and nitroisocitrate bound | journal = Biochemistry | volume = 31 | issue = 10 | pages = 2735–48 | date = Mar 1992 | pmid = 1547214 | doi = 10.1021/bi00125a014 }} 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.

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 number|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. Fluoroacetate, in the citric acid cycle, is converted to fluorocitrate by citrate synthase. Fluorocitrate competitively inhibits aconitase halting the citric acid cycle.{{cite journal | vauthors = Morrison JF, Peters RA | title = Biochemistry of fluoroacetate poisoning: the effect of fluorocitrate on purified aconitase | journal = Biochem. J. | volume = 58| issue = 3| pages = 473–9|date=November 1954| doi = 10.1042/bj0580473 | pmid = 13208639| pmc = 1269923 }} The iron sulfur cluster is highly sensitive to oxidation by superoxide.{{cite book |pages=9–23 |doi=10.1016/S0076-6879(02)49317-2 |chapter=Aconitase: Sensitive target and measure of superoxide |title=Superoxide Dismutase |series=Methods in Enzymology |year=2002 |last1=Gardner |first1=Paul R. |isbn=978-0-12-182252-1 |volume=349|pmid=11912933 }}

File:Arrow Pushing Aconitase Final draft.tif

File:Citrate Zoom Final.png

Aconitase employs a dehydration-hydration mechanism. The catalytic residues involved are His-101 and Ser-642.{{cite web|url=http://web.ku.edu/~crystal/taksnotes/Biol_638/notes/chp_16.pdf |title=Chapter 16: Citric Acid Cycle |author=Takusagawa F |work=Takusagawa’s Note |publisher=The University of Kansas |access-date=2011-07-10 |url-status=dead |archive-url=https://web.archive.org/web/20120324072437/http://web.ku.edu/~crystal/taksnotes/Biol_638/notes/chp_16.pdf |archive-date=2012-03-24 }} 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-called cis-aconitate intermediate (the two carboxyl groups on the double bond are cis).{{cite journal | vauthors = Han D, Canali R, Garcia J, Aguilera R, Gallaher TK, Cadenas E | title = Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione | journal = Biochemistry | volume = 44 | issue = 36 | pages = 11986–96 | date = Sep 2005 | pmid = 16142896 | doi = 10.1021/bi0509393 }} The carbon atom from which the hydrogen is removed is the one that came from oxaloacetate in the previous step of the citric acid cycle, not the one that came from acetyl CoA, even though these two carbons are equivalent except that one is "pro-R" and the other "pro-S" (see Prochirality).{{cite book|author=Lubert Stryer|author-link=Lubert Stryer|title=Biochemistry|date=1981 |edition=2nd|title-link=Biochemistry (Stryer)|pages=295–296}}{{rp|393}} At this point, the intermediate is rotated 180°. This rotation is referred to as a "flip."{{cite journal | vauthors = Beinert H, Kennedy MC, Stout CD | title = Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein | journal = Chemical Reviews | volume = 96 | issue = 7 | pages = 2335–2374 | date = Nov 1996 | pmid = 11848830 | doi = 10.1021/cr950040z | url = http://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf | access-date = 2011-05-16 | archive-url = https://web.archive.org/web/20110811075307/http://alchemy.chem.uwm.edu/classes/chem601/Handouts/beinert.pdf | archive-date = 2011-08-11 | url-status = dead }} Because of this flip, the intermediate is said to move from a "citrate mode" to a "isocitrate mode."{{cite journal | vauthors = Lauble H, Stout CD | title = Steric and conformational features of the aconitase mechanism | journal = Proteins | volume = 22 | issue = 1 | pages = 1–11 | date = May 1995 | pmid = 7675781 | doi = 10.1002/prot.340220102 | s2cid = 43006515 }}

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. This rate-limiting step ensures that the right stereochemistry, specifically (2R,3S), is formed in the final product.{{cite web | url = http://metallo.scripps.edu/PROMISE/ACONITASE.html | title = Aconitase family | date = 1999-02-02 | work = The Prosthetic groups and Metal Ions in Protein Active Sites Database Version 2.0 | publisher = The University of Leeds | access-date = 2011-07-10 | archive-url = https://web.archive.org/web/20110608223412/http://metallo.scripps.edu/PROMISE/ACONITASE.html | archive-date = 2011-06-08 | url-status = dead }} Another hypothesis is that cis-aconitate stays bound to the enzyme while it flips from the citrate to the isocitrate mode.

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

File:Isocitrate Zoom Final.png

Aconitases are expressed in bacteria to humans. In citrus fruits, a reduction of the activity of the mitochondrial aconitases likely leads to the buildup of citric acid, which is then stored in vacuoles.{{cite journal |last1=Degu |first1=Asfaw |last2=Hatew |first2=Bayissa |last3=Nunes-Nesi |first3=Adriano |last4=Shlizerman |first4=Ludmila |last5=Zur |first5=Naftali |last6=Katz |first6=Ehud |last7=Fernie |first7=Alisdair R. |last8=Blumwald |first8=Eduardo |last9=Sadka |first9=Avi |title=Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis |journal=Planta |date=September 2011 |volume=234 |issue=3 |pages=501–513 |doi=10.1007/s00425-011-1411-2}} 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. Humans express the following two aconitase isozymes:

{{TCACycle WP78|highlight=Aconitase}}

{{reflist|2}}

{{refbegin}}

• {{cite journal | vauthors = Frishman D, Hentze MW | title = Conservation of aconitase residues revealed by multiple sequence analysis. Implications for structure/function relationships | journal = European Journal of Biochemistry | volume = 239 | issue = 1 | pages = 197–200 | date = Jul 1996 | pmid = 8706708 | doi = 10.1111/j.1432-1033.1996.0197u.x | doi-access = free }}

{{refend}}

• {{MeshName|Aconitase}}
• {{Proteopedia|Aconitase}} - the Aconitase structure in interactive 3D

{{Citric acid cycle enzymes}}

{{Mitochondrial enzymes}}

{{Carbon-oxygen lyases}}

{{Enzymes}}

{{Portal bar|Biology|border=no}}

Category:EC 4.2.1

Category:Iron–sulfur proteins

Category:Moonlighting proteins