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Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay

1981; Elsevier BV; Volume: 208; Issue: 1 Linguagem: Inglês

10.1016/0003-9861(81)90140-5

1096-0384

Richard B. Westkaemper, Robert P. Hanzlik,

Metabolism and Genetic Disorders

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 (H2OD2O) 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.