7-Dehydrocholesterol 5,6 beta-oxide as a mechanism-based inhibitor of microsomal cholesterol oxide hydrolase

7-Dehydrocholesterol 5,6 beta-oxide covalently modifies and inactivates the rat liver microsomal enzyme cholesterol oxide hydrolase. The covalent modification is presumed to occur at the active site of the enzyme since 5,6 alpha-iminocholestanol, a potent competitive inhibitor of the enzyme, blocks incorporation of 3-[3H]-7-dehydrocholesterol 5,6 beta-oxide into the protein. Kinetics of the inactivation were measured both by following the loss of catalytic activity and by monitoring incorporation of 3-[3H]-7-dehydrocholesterol 5,6 beta-oxide into microsomal protein. Both the loss of catalytic activity and the incorporation of label followed first order kinetics. Linear plots of the reciprocal of the pseudo-first order rate constants for the loss of catalytic activity and for the incorporation of radioactivity versus reciprocal of inhibitor concentrations indicated saturation kinetics. The kinetic parameter kinac is found to be (2.83 +/- 0.43)10(-3) s-1 measured either by incorporation of tritium (300 mM potassium phosphate buffer, pH 8.0, 2.4 mg of microsomal protein/ml at 37 degrees C) or by the loss of catalytic activity (300 mM potassium phosphate buffer, pH 7.5, 0.99 mg of microsomal protein/ml at 37 degrees C). Unlike xenobiotic microsomal epoxide hydrolase (EC 3.3.2.3) which is not inactivated or inhibited by 7-dehydrocholesterol 5,6 beta-oxide, cholesterol oxide hydrolase appears to hydrolyze cholesterol oxides via a positively charged transition state.