Fourier-Transform Ion Cyclotron Resonance Mass Spectrometric Evidence for the Formation of .alpha.-Chloroenethiolates and Thioketenes from Chloroalkene-Derived, Cytotoxic 4-Thiaalkanoates

Zhang, Tian‐Lan; Wang, Liping; Hashmi, M.; Anders, M.W.; Thorpe, Colin; Ridge, D. P.

doi:10.1021/tx00049a002

Cited by 12 publications

(8 citation statements)

References 23 publications

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“…This reaction was previously described with simpler 4-thia fatty acyl-CoA analogues (20). Model reactions, both in organic solvents and in the gas phase, provide support for the formation of the 1,2-dichloroethenethiolate anion (compound 5) as an immediate product of this elimination reaction (21). These studies also provided evidence for chlorothioketene formation (compound 7) as one route in the decomposition of compound 5 (21,22).…”

supporting

confidence: 57%

“…Model reactions, both in organic solvents and in the gas phase, provide support for the formation of the 1,2-dichloroethenethiolate anion (compound 5) as an immediate product of this elimination reaction (). These studies also provided evidence for chlorothioketene formation (compound 7) as one route in the decomposition of compound 5 ( , ). The thionoacyl chloride (compound 8) is another likely participant in the decomposition of 1,2-dichloroethenethiolate ( − ).…”

mentioning

confidence: 85%

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Elimination Reactions in the Medium-Chain Acyl-CoA Dehydrogenase: Bioactivation of Cytotoxic 4-Thiaalkanoic Acids

et al. 1998

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A range of 4-thiaacyl-CoA derivatives has been synthesized to study the bioactivation of cytotoxic fatty acids by the mitochondrial medium-chain acyl-CoA dehydrogenase and the peroxisomal acyl-CoA oxidase. Both enzymes catalyze alpha-proton abstraction from normal acyl-CoA substrates with elimination of a beta-hydride equivalent to the FAD prosthetic group. In competition with this oxidation reaction, 4-thiaacyl-CoA thioesters undergo dehydrogenase-catalyzed beta-elimination, providing that the corresponding thiolates are sufficiently good leaving groups and can be accommodated by the active site of the enzyme. Thus, the dehydrogenase catalyzes the elimination of 2-mercaptobenzothiazole and 4-nitrothiophenolate from 4-(2-benzothiazole)-4-thiabutanoyl-CoA and 4-(4-nitrophenyl)-4-thiabutanoyl-CoA, respectively. However, the 2,4-dinitrophenyl-analogue appears too bulky and the unsubstituted thiophenyl-derivative is insufficiently activated for significant elimination. Molecular modeling shows that steric interference from the flavin ring dictates a syn rather than an anti elimination. Acryloyl-CoA, the other product of 4-thiaacyl-CoA elimination reactions, is not a significant inactivator of the medium-chain dehydrogenase. In contrast, the irreversible inactivation observed during beta-elimination using 5,6-dichloro-4-thia-5-hexenoyl-CoA (DCTH-CoA), 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA), and 6-chloro-5,5,6-trifluoro-4-thiahexanoyl-CoA (CTFTH-CoA) is caused by release of cytotoxic thiolate products within the active site of the dehydrogenase. The double bond between C5 and C6 found in the vinylic analogues DCTH- and DCTFTH-CoA is not essential for enzyme inactivation, although CTFTH-CoA is a weaker inhibitor of the dehydrogenase. Mechanism-based inactivation with CTFTH-CoA requires elimination, is unaffected by exogenous nucleophiles, and is strongly protected by octanoyl-CoA. The peroxisomal acyl-CoA oxidase efficiently oxidizes 4-thiaacyl-CoA analogues, but is only rapidly inactivated by DCTFTH-CoA. The variable ratio of elimination to oxidation observed for DCTH-, DCTFTH-, and CTFTH-CoA may influence the metabolism of the corresponding cytotoxic alkanoic acids in vivo.

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confidence: 57%

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Elimination Reactions in the Medium-Chain Acyl-CoA Dehydrogenase: Bioactivation of Cytotoxic 4-Thiaalkanoic Acids

et al. 1998

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“…InactiVation of the ECH by DCTFTH-CoA. Reaction of DCTFTH-CoA (10) with the enoyl-CoA hydratase (Scheme 4) would be expected to generate 1,2-dichloro-3,3,3-trifluoro-1-propenethiolate ( 11) and acryloyl-CoA (6), by analogy with earlier studies with BTTB and DCTH thioesters (27,28,33).…”

Section: Resultsmentioning

confidence: 78%

Novel Inactivation of Enoyl-CoA Hydratase via β-Elimination of 5,6-Dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA

Baker-Malcolm¹,

Lantz²,

Anderson³

et al. 2000

Biochemistry

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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.

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“…A completely different type of mechanism‐based inactivation of ACADs is provided by compounds A and B in Fig. 7[102,103]. 5,6‐Dichloro‐4‐thia‐5‐hexenoyl‐CoA (compound A) is a prototype of these compounds and is activated approximately one in every five turnovers.…”

Section: Protein‐directed Inactivatorsmentioning

confidence: 99%

Acyl‐CoA dehydrogenases

Ghisla

Thorpe

2004

European Journal of Biochemistry

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307

297

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Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the a,b-dehydrogenation of fatty acid acyl-CoA conjugates. While they differ widely in their specificity, they share the same basic chemical mechanism of a,b-dehydrogenation. Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms. Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein. The mechanism of a,bdehydrogenation is viewed as a process in which the substrate aC-H and bC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO -as an H + , the second being transferred as a hydride to the flavin N(5) position. Hereby the pK a of the substrate aC-H is lowered from > 20 to % 8 by the effect of specific hydrogen bonds. Concomitantly, the pK a of Glu376-COO -is also raised to 8-9 due to the decrease in polarity brought about by substrate binding. The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates. A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein. These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases.

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Fourier-Transform Ion Cyclotron Resonance Mass Spectrometric Evidence for the Formation of .alpha.-Chloroenethiolates and Thioketenes from Chloroalkene-Derived, Cytotoxic 4-Thiaalkanoates

Cited by 12 publications

References 23 publications

Elimination Reactions in the Medium-Chain Acyl-CoA Dehydrogenase: Bioactivation of Cytotoxic 4-Thiaalkanoic Acids

Elimination Reactions in the Medium-Chain Acyl-CoA Dehydrogenase: Bioactivation of Cytotoxic 4-Thiaalkanoic Acids

Novel Inactivation of Enoyl-CoA Hydratase via β-Elimination of 5,6-Dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA

Acyl‐CoA dehydrogenases

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