Aconitase

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Aconitase is also called aconitic acid hydratase. In the tricarboxylic acid cycle, a coenzyme that catalyzes the conversion between citric acid and isocitrate. Under equilibrium conditions, the ratio of the three substances is 91:3:6 (aconitic acid enzyme balance). It is widely distributed in animals, plants and microorganisms, and the content in liver, kidney, muscle, and prostate is particularly high. It was once thought that it is the same enzyme as Fumarase, and it is also believed that the above-mentioned reaction involves two different enzymes, which are now clearly catalyzed by a single enzyme. Enzyme activity can be strongly inhibited by transaconitic acid and fluorinated citric acid. The reaction mechanism of this enzyme lies in the highly specific three-point binding through the iron-citrate complex, and the binding to the active site of the enzyme has attracted more attention.

Structure

The aconitase shown in the structure on the right edge of the 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 interaction with the [3Fe-4S] cluster, but the active site is composed of residues from all four domains, including the larger C-terminal domain. Fe-S clusters and SO42-anions are also located at the active site. After activating the enzyme, it will acquire an extra iron atom, thus forming a [4Fe-4S] cluster. However, the structure of the remaining enzymes hardly changed. The conservative atoms between the two forms are basically in the same position, with a difference of 0.1 angstroms.

Figure 1. Aconitase family.

Biochemical properties

Similar to animals, aconitase has at least two isoenzymes of cytoplasm and mitochondria. But Bellis et al. found in pumpkin cotyledons that in addition to isoenzyme Ⅰ (ACO Ⅰ) and isoenzyme Ⅱ (ACO Ⅱ), there may also be isoenzyme Ⅲ (ACO Ⅲ). They further studied and found that cis-aconitic acid enzymes, especially mitochondrial isoenzymes, are relatively unstable enzymes, which are easily inactivated during the separation and purification process; the obvious difference from animal bodies is that they are derived from the same plant material. The subcellular distribution of aconitase isoenzymes is not easy to be separated due to their similar isoelectric points (pI). Therefore, the separation and purification of plant aconitase and its isoenzymes are relatively lagging behind animal bodies. Related research. In addition, some of the enzymatic properties of cis-aconitic acid are also different due to different plant materials.

Sensitivity to reactive oxygen species

Animal and human aconitase are very sensitive to ROS, which may be related to the presence of 4Fe-4S which is easily oxidized in its active site. For example, human mitochondrial aconitase contains 4Fe-4S and becomes a redox sensor that is very sensitive to ROS, while O-2 inactivates it, and at the same time causes the level of H2O2 and free Fe²⁺ in the mitochondria to increase, thereby inducing the generation of OH. The large amount of ROS produced in the process of adversity and aging can irreversibly inactivate cisconitase, and this inactivation is related to the carboxylation reaction caused by ROS at the active site of cisconitase and depends on metal ions and has a site. The point-specific oxidation is closely related.

Conclusions

So far, although people have gained a certain understanding of the characteristics and functions of aconitase, the regulation of NO, ROS and other active molecules on this enzyme has only been confirmed in a few plant materials such as pumpkin and tobacco.