Mechanism of soluble epoxide hydrolase: Formation of an α-hydroxy ester-enzyme intermediate through Asp-333

Babak Borhan, A. Daniel Jones, Franck Pinot, David F. Grant, Mark J. Kurth, Bruce D. Hammock

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Abstract

18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H2 18O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 18O. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an α-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.

Original languageEnglish (US)
Pages (from-to)26923-26930
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number45
StatePublished - Nov 10 1995

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ASJC Scopus subject areas

  • Biochemistry

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Borhan, B., Jones, A. D., Pinot, F., Grant, D. F., Kurth, M. J., & Hammock, B. D. (1995). Mechanism of soluble epoxide hydrolase: Formation of an α-hydroxy ester-enzyme intermediate through Asp-333. Journal of Biological Chemistry, 270(45), 26923-26930.