Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay |
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Authors: | Richard B Westkaemper Robert P Hanzlik |
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Institution: | Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045 U.S.A. |
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Abstract: | A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the Km and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; Km remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D2O the solvent isotope effect on V () is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of 7-2H]PNSO and a 4% normal isotope effect for the hydration of 8-2H2]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism. |
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