MECHANISM OF SOLUBLE EPOXIDE HYDROLASE - FORMATION OF AN ALPHA-HYDROXY ESTER-ENZYME INTERMEDIATE THROUGH ASP-333

O-18-labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and c is-9,10-epoxystearic acid were synthesized and used to determine the r egioselectivity of sEH. The nucleophilic nature of sEH catalysis was d emonstrated by comparing the enzymatic and nonenzymatic hydrolysis pro ducts of tDPPO. The results from single turnover experiments with grea ter or equal molar equivalents of sEH:substrate were consistent with t he existence of a stable intermediate formed by a nucleophilic amino a cid attacking the epoxide group. Tryptic digestion of sEH previously s ubjected to multiple turnovers with tDPPO in (H2O)-O-18 resulted in th e isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustra ted the incorporation of O-18. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attach by Asp-333 initiates enzymatic activity, leading to the for mation of an alpha-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl enzyme occurs by the addition of an activated water to the carbon yl carbon of the ester bond, after which the resultant tetrahedral int ermediate collapses, yielding the active enzyme and the diol product.