Submitted to: Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2005
Publication Date: 8/19/2005
Citation: Tran, K.L., Aronov, P.A., Tanaka, H., Newman, J.W., Hammock, B.D., Morisseau, C. Lipid sulfates and sulfonates are allosteric competitive inhibitors of the n-terminal phosphatase activity of the mammalian soluble epoxide hydrolase. Journal of Biochemistry. 44:12179-12187, 2005. Interpretive Summary: The soluble epoxide hydrolase is an enzyme with important roles in the regulation of vascular and renal function. It was recently discovered that this enzyme has two independent and functional catalytic sites. One of these is positioned in the C-terminal domain of the protein and is responsible for the well studied epoxide hydrolase activity of the protein. The other catalytic site is positioned in the N-terminal domain of the protein and has small molecule phosphatase activity. The removal of a phosphate from molecules changes there properties greatly, and is routinely used as a chemical switch to regulate biological processes. In this study we developed an efficient enzyme assay and the first potent inhibitors of this phosphatase activity. We then used these procedures to evaluate additional naturally occurring small molecule phosphates as substrates and discovered that isoprenyl phosphates are excellent substrates, suggesting a link between the soluble epoxide hydrolase phosphatase activity and the regulation of cholesterol metabolism and inflammation. Regardless, the biochemical tools and understanding reported will facilitate an accelerated exploration of the biological function of the other half of this important enzyme.
Technical Abstract: The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the Nterminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-Phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-Phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate and phosphonate lipids as novel potent inhibitors of Nterm-Phos. These compounds are allosteric competitive inhibitors with KI in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that poly-isoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a non-competitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.