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

被引:8
作者
Baker-Malcolm, JF
Lantz, M
Anderson, VE
Thorpe, C [1 ]
机构
[1] Univ Delaware, Dept Chem & Biochem, Newark, DE 19716 USA
[2] Case Western Reserve Univ, Dept Biochem, Cleveland, OH 44106 USA
关键词
D O I
10.1021/bi0010165
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
5,6-Dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA) is an analogue of a class of cytotoxic 4-thiaacyl-CoA thioesters that can undergo a beta-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-elimination of the reactive thiolate moiety [Baker-Malcolm, J, F., Haeffner-Gormley, L., Wang, L., Anders, M. W., and Thorpe, C, (1998) Biochemistry 37, 1383-1393]. This paper shows that DCTFTH-CoA can be directly bioactivated by the enoyl-CoA hydratase (ECH) with the release of 1,2-dichloro-3,3,3-trifluoro-1-propenethiolate and acryloyl-CoA. In the absence of competing exogensus trapping agents, DCTFTH-CoA effects rapid and irreversible loss of hydratase activity. The inactivator is particularly effective at pH 9.0, with a stoichiometry approaching 1 mol of DCTFTH-CoA per enzyme subunit. Modification is associated with a new protein-bound chromophore at 360 nm and an increase 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-CoA. 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, including small thiol nucleophiles, various proteins, and even phospholipid bilayers, exert strong protection against modification of ECH. Peptide mapping, thiol titrations, UV-vis spectrophotometry, and mass spectrometry show that inactivation involves the covalent modification of Cys62 and/or Cys111 of the recombinant rat liver ECH. These data suggest that enoyl-CoA hydratase is an important enzyme in the bioactivation of DCTFTH-CoA, in a pathway which does not require involvement of the medium-chain acyl-CoA dehydrogenase.
引用
收藏
页码:12007 / 12018
页数:12
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