Novel inactivation of enoyl-CoA hydratase via beta-elimination of 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA

5,6-Dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA) is an anal ogue of a class of cytotoxic 4-thiaacyl-CoA thioesters that can undergo a b eta-elimination reaction to form highly unstable thiolate fragments, which yield electrophilic thioketene or thionoacyl halide species. Previous work demonstrated that the medium-chain acyl-CoA dehydrogenase both bioactivates and is inhibited by these CoA thioesters through enzyme-catalyzed beta-eli mination of the reactive thiolate moiety [Baker-Malcolm, J, F., Haeffner-Go rmley, L., Wang, L., Anders, M. W., and Thorpe, C, (1998) Biochemistry 37, 1383-1393]. This paper shows that DCTFTH-CoA can be directly bioactivated b y the enoyl-CoA hydratase (ECH) with the release of 1,2-dichloro-3,3,3-trif luoro-1-propenethiolate and acryloyl-CoA. In the absence of competing exoge nsus trapping agents, DCTFTH-CoA effects rapid and irreversible loss of hyd ratase activity. The inactivator is particularly effective at pH 9.0, with a stoichiometry approaching 1 mol of DCTFTH-CoA per enzyme subunit. Modific ation is associated with a new protein-bound chromophore at 360 nm and an i ncrease in mass of 89 +/- 5 per subunit. Surprisingly, ECH exhibiting less than 2% residual hydratase activity retains essentially 100% beta-eliminase activity and continues to generate reactive thiolate species from DCTFTH-C oA. This leads to progressive derivatization of the enzyme with additional UV absorbance, covalent cross-linking of subunits, and an eventual complete loss of beta-eliminase activity. A range of exogenous trapping agents, inc luding small thiol nucleophiles, various proteins, and even phospholipid bi layers, exert strong protection against modification of ECH. Peptide mappin g, thiol titrations, UV-vis spectrophotometry, and mass spectrometry show t hat inactivation involves the covalent modification of Cys62 and/or Cys111 of the recombinant rat liver ECH. These data suggest that enoyl-CoA hydrata se is an important enzyme in the bioactivation of DCTFTH-CoA, in a pathway which does not require involvement of the medium-chain acyl-CoA dehydrogena se.