Sandbox Reserved 714: Difference between revisions
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The Human soluble Epoxide hydrolase is a protein of 555 residues. In vivo, it exists under the form of a homodimer, with a monomeric unit of 62,5 kDa. Each subunit has <scene name='Sandbox_Reserved_714/Catalytic_domains/5'>two catalytic domains</scene>, linked by a proline-rich section. | The Human soluble Epoxide hydrolase is a protein of 555 residues. In vivo, it exists under the form of a homodimer, with a monomeric unit of 62,5 kDa. Each subunit has <scene name='Sandbox_Reserved_714/Catalytic_domains/5'>two catalytic domains</scene>, linked by a proline-rich section. | ||
The secondary structure of this enzyme is made of beta-sheets (16 strands) and alpha-helices and a few 310-helices (34 helices), which form the two pockets of the active sites, in the C-term domain and the N-term domain, in which the substrates (lipid-phosphates and ions for the N-term domain, and epoxides for the C-term domain) can bind. The 3D dimensions of the enzyme are the following : a = 92.55 Å, b = 92.55 Å, c = 244.64 Å. | |||
The quaternary structure is called a domain-swapped dimer. The C-ter domain adopts an α/β-hydrolase fold, corresponding to its catalytic activity. Remarkably, the N-term domain has an α/β fold which is similar to HAD (Haloacid Dehalogenase) family. This N-term domain has no dehalogenase activity but a phosphatase one. | |||
The N-terminal domain has specific features that facilitate the binding of a lipid substrate. There are <scene name='Sandbox_Reserved_714/Nter_cleft-tunnel-activesite/2'>three sites</scene> that ensure the proper positioning of the substrate. First, a hydrophobic cleft of about 25 Å long is situated near the N-term core so that one of the two ends of the aliphatic substrate is near the interface between the two domains N-term and C-term (proline-rich linker). Secondly, a hydrophobic tunnel (about 14 Å long) binds the aliphatic chain of the substrate and allows the second end to be in the active site. The active site is a negatively charged pocket of about 15 Å deep, and contains a Mg<sup>2+</sup> cation necessary to the catalytic function. | The N-terminal domain has specific features that facilitate the binding of a lipid substrate. There are <scene name='Sandbox_Reserved_714/Nter_cleft-tunnel-activesite/2'>three sites</scene> that ensure the proper positioning of the substrate. First, a hydrophobic cleft of about 25 Å long is situated near the N-term core so that one of the two ends of the aliphatic substrate is near the interface between the two domains N-term and C-term (proline-rich linker). Secondly, a hydrophobic tunnel (about 14 Å long) binds the aliphatic chain of the substrate and allows the second end to be in the active site. The active site is a negatively charged pocket of about 15 Å deep, and contains a Mg<sup>2+</sup> cation necessary to the catalytic function. | ||
The aminoacids which are involved in the active site of the N-term domain are the Asp9, Asp11 and Asp185 basic aminoacids which can bind with a Mg<sup>2+</sup> ion each, which is necessary to permit the substrate binding. These residues are characteristic of a significant part of phosphatases and phosphonatases. There is also a modified lysine, the Lys43, which has an acetyl group on its N6. | |||
The aminoacids which are involved in the active site of the C-term domain can be divided in two groups : the binding aminoacids and the catalytic aminoacids. | |||
The His239, Tyr241, Arg249 and Glu298 are involved in the binding of the substrate, the Hexaethylene Glycol (P6G). | |||
The Asp335, Asp496, Tyr466 and His524 are the catalytic aminoacids of the active site. Asp335 can lead nucleophilic additions, Tyr466 is a proton donor and His524 is a proton acceptor. | |||
== Mechanism == | == Mechanism == | ||
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=== C-terminal domain === | === C-terminal domain === | ||
The C-terminal domain is called Cytosolic epoxide hydrolase 2: it catalyzes the trans-addition of water to epoxides in order to product glycols<ref>PMID:15822179</ref>. | The C-terminal domain is called Cytosolic epoxide hydrolase 2: it catalyzes the trans-addition of water to epoxides in order to product glycols <ref name="EH">PMID:15822179</ref>. | ||
The corresponding reaction equation is the following: | The corresponding reaction equation is the following: | ||
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== Inhibitors == | == Inhibitors == | ||
As many enzymes, the human soluble epoxide hydrolase can be inhibited by some inhibitors, causing a loss of activity for the enzyme. | |||
The sEH can be inhibited by some metal ions such as Zn<sup>2+</sup>, Cu<sup>2+</sup>, Hg<sup>2+</sup> <ref name="EH" />. It is a noncompetitive inhibition: the metal ion binding to the enzyme reduces its activity without affecting directly the substrate binding, so that the V<sub>max</sub> decreases but the K<sub>m</sub> remains unchanged. It may be that those metal ions replace the Mg<sup>2+</sup> in the N-term active site of the enzyme subunits, which can result in some conformation changes, but this is only an hypothesis. | |||
However, there are also some chemical inhibitors that inhibit the sEH. They are 1-3 disubstitued ureas, carbamates and amides, which are stable inhibitors for the sEH. | |||
Those chemical inhibitors are competitive inhibitors, which can bind in the active site of the enzyme in place of the right substrate, which graphically can be noticed by a K<sub>m</sub> increase, whereas the V<sub>max</sub> remains unchanged. In fact, according to crystal structures, urea can set up hydrogen bonds and salt bridges with some residues of the sEH active site, which ends up in an “enzyme-substrate”-like complex. Nevertheless, it is also important to notice that those inhibitor must have hydrophobic sidechains in order to fit in the hydrophobic zone of the catalytic site. | |||
</StructureSection> | </StructureSection> | ||