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 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 exogenous 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.
Baker-Malcolm
J F
JF
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Lantz
M
M
Anderson
V E
VE
Thorpe
C
C
D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004746:Enoyl-CoA Hydratase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D008024:Ligands; D008930:Mitochondria, Liver / Q000201:enzymology; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
C414551:5, 6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA; D000214:Acyl Coenzyme A; D008024:Ligands; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D004247:DNA; C010701:crotonyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D004746:Enoyl-CoA Hydratase