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Enzyme Mechanism
Aconitase employs a dehydration-hydration mechanism. The catalytic residues involved are His-101 and Ser-642. 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. At this point, the intermediate is rotated 180º. This rotation is referred to as a "flip." Because of this flip, the intermediate is said to move from a "citrate mode" to a "isocitrate mode."

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-liming step ensures that the right stereochemistry, specifically (2R,3S), is formed in the final product. 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 to prime it to attack at C2, and the protonated serine is deprotonated by the cis-aconitate double bond to complete the hydration, producing isocitrate.



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. 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 SO42- anion also reside in the active site. When the enzyme is activated, it gains an addition iron atom, creating a [4Fe-4S] cluster. 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.

Disease Relevance
A serious ailment associated with aconitase is known as aconitase deficiency. 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. The main symptoms are myopathy and exercise intolerance; physical strain is lethal for some patients because it can lead to circulatory shock. There are no known treatments for aconitase deficiency.

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. 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. This deviance from homoestasis causes FRDA, a neurodegenerative disease for which no effective treatments have been found.

Finally, aconitase is thought to be associated with diabetes. Although the exact connection is still being determined, multiple theories exist. In a study of organs from mice with alloxan diabetes (experimentally induced diabetes ) and genetic diabetes, lower aconitase activity was found to decrease the rate of metabolic reactions involving citrate, pyruvate, and malate. In addition, citrate concentration was observed to be abnormally high. Since these abnormal data were found in diabetic mice, the study concluded that low aconitase activity is correlated with genetic and alloxan diabetes. 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. That is, it converts isocitrate back to cis-aconitate more rapidly than usual, but the forward reaction proceeds at the usual rate. This imbalance may contribute to disrupted metabolism in diabetics.