PMID- 11009615
OWN - NLM
STAT- MEDLINE
DCOM- 20001027
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 39
IP  - 39
DP  - 2000 Oct 3
TI  - Novel inactivation of enoyl-CoA hydratase via beta-elimination of 5, 
      6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA.
PG  - 12007-18
AB  - 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.
FAU - Baker-Malcolm, J F
AU  - Baker-Malcolm JF
AD  - Department of Chemistry and Biochemistry, University of Delaware, Newark, 
      Delaware 19716, USA.
FAU - Lantz, M
AU  - Lantz M
FAU - Anderson, V E
AU  - Anderson VE
FAU - Thorpe, C
AU  - Thorpe C
LA  - eng
GR  - 1-T32-GM-08550/GM/NIGMS NIH HHS/United States
GR  - GM26643/GM/NIGMS NIH HHS/United States
GR  - GM36562/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (5, 6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA)
RN  - 0 (Acyl Coenzyme A)
RN  - 0 (Ligands)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Sulfhydryl Compounds)
RN  - 9007-49-2 (DNA)
RN  - 992-67-6 (crotonyl-coenzyme A)
RN  - EC 1.3.- (Acyl-CoA Dehydrogenases)
RN  - EC 1.3.8.7 (Acyl-CoA Dehydrogenase)
RN  - EC 4.2.1.17 (Enoyl-CoA Hydratase)
SB  - IM
MH  - Acyl Coenzyme A/*chemistry/metabolism
MH  - Acyl-CoA Dehydrogenase
MH  - Acyl-CoA Dehydrogenases/chemistry/metabolism
MH  - Animals
MH  - Binding Sites
MH  - Catalysis
MH  - DNA/chemistry
MH  - Enoyl-CoA Hydratase/*antagonists & inhibitors/*chemistry/genetics/metabolism
MH  - Enzyme Activation
MH  - Ligands
MH  - Mitochondria, Liver/enzymology
MH  - Rats
MH  - Recombinant Proteins/antagonists & inhibitors/chemistry/metabolism
MH  - Sulfhydryl Compounds/chemistry/metabolism
EDAT- 2000/09/29 11:00
MHDA- 2001/02/28 10:01
CRDT- 2000/09/29 11:00
PHST- 2000/09/29 11:00 [pubmed]
PHST- 2001/02/28 10:01 [medline]
PHST- 2000/09/29 11:00 [entrez]
AID - bi0010165 [pii]
AID - 10.1021/bi0010165 [doi]
PST - ppublish
SO  - Biochemistry. 2000 Oct 3;39(39):12007-18. doi: 10.1021/bi0010165.