General structure
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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 two catalytic domains, linked by a proline-rich section.
Mechanism
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[1].
The corresponding reaction equation is the following:
Epoxide + H2O ↔ Glycol
The active site is made of five residues. The 3D structure of this active site is maintained by hydrogen bonds, including those created by D496. The two tyrosines (Y383 and Y466) assist the proper positioning of the substrate by polarizing it, thanks to their hydroxyl groups. D335 acts as a nucleophilic acid. Finally, H524 plays the role of a base in order to release the final product.
The reaction proceeds in two steps, including the formation of a covalent intermediate.
First, the substrate (epoxide) is accepted in the active site and its oxygen forms hydrogen bonds with Y383 and Y466. The oxygen of D335 attacks one of the two carbons included in the epoxide function. As a result, the oxygen of the subtrate takes the hydrogen of the hydroxyl function of Y466: the covalent intermediate is formed, and linked to D335.
Then, the oxygen negatively charged belonging to the lateral chain of Y383 attacks the hydrogen of H524. After that, a water molecule enters the active site. The hydroxyl of D335 is therefore renewed, the diol product is created and released. The active site is available for a new catalytic cycle.
N-terminal domain
The N-terminal domain is responsible of the Mg2+ dependant hydrolysis of dihydroxy lipid phosphates [2]. Indeed, the aliphatic substrate binds the protein on its hydrophobic tunnel, as it has been described previously. The specificity of this enzyme has been tested for several lipid molecules, and the best substrate found is the monophosphate of dihydroxy stearic acid (threo-9/10-phosphonoxy-hydroxy-octadecanoic acid) [3]. Indeed, the catalytic values for this substrate are Km = 21 +/- 0.3 μ�M, VMax = 338 �+/- 12 nmol.�min�-1�.mg�-1, and kcat =� 0.35 +/-� 0.01 s-�1.
In the example of this substrate, the reaction follows this equation:
9/10-phosphonoxy-hydroxy-octadecanoate + H2O ↔ 9/10-dihydroxy-octadecanoate + phosphate
Its active site contains several conserved aspartates in phosphatases and phosphonatases: D9, D11, D184 and D185. This enzymatic activity is Mg2+ dependant, because the structure of the active site is in its optimal conformation when the cation makes coordination interactions. When the catalytic activity of the N-term domain is available, Magnesium is octahedrally coordinated with the four aspartates, one water molecule and the phosphate belonging to the substrate. We can note that Mg2+ is not directly involved in the catalytic mechanism, and all its interactions with the active site remain during the hydrolysis. Its single role consists in maintaining the three-dimensional structure of the active site.
First, the oxygen on the lateral chain of D9 attacks the phosphate. After the addition of a proton H+, the product with its two hydroxyl functions is released, while the phosphate is still linked to D9. Then, a waters molecule binds the phosphate, breaking its bond with the aspartate. Therefore the phosphate can be finally released and the active site can accept a lipid again and start a new catalytic cycle.
Inhibitors
- ↑ Morisseau C, Hammock BD. Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu Rev Pharmacol Toxicol. 2005;45:311-33. PMID:15822179 doi:10.1146/annurev.pharmtox.45.120403.095920
- ↑ Gomez GA, Morisseau C, Hammock BD, Christianson DW. Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis. Biochemistry. 2004 Apr 27;43(16):4716-23. PMID:15096040 doi:10.1021/bi036189j
- ↑ Newman JW, Morisseau C, Harris TR, Hammock BD. The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1558-63. Epub 2003 Feb 6. PMID:12574510 doi:10.1073/pnas.0437724100
