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

Abstract

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.

Role of Glutamate 144 and Glutamate 164 in the Catalytic Mechanism of Enoyl-CoA Hydratase

“…Steady-state kinetics revealed a k cat of 0.18 s -1 and a K m of 35 µM for wild-type enoyl-CoA hydratase. This contrasts with a k cat of 0.12 s -1 and a K m of 6 µM for acyl-CoA dehydrogenase (20). Thus, enoyl-CoA hydratase and acyl-CoA dehydrogenase have similar abilities to eliminate 2-mercaptobenzothiazole from BTTB-CoA.…”

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

“…The following acids were synthesized as described previously: 5,6-dichloro-4-thia-5-hexenoic acid (DCTH; 42), 6-chloro-5,5,6-trifluoro-4-thiahexanoic acid (CTFTH; 30), 4-(2-benzothiazole)-4thiabutanoic acid (BTTB; 30), 4-(4-nitrophenyl)-4-thiabutanoic acid (NPTB; 28), and 4-(2,4-dinitrophenyl)-4-thiabutanoic acid (DNPTB; 28). 5,6-Dichloro-7,7,7-trifluoro-4-thia-5heptenoic acid (DCTFTH) was synthesized as described previously (28) with the following modifications. 3-Mercaptopropionic acid (18.8 mmol) was added dropwise to a suspension of lithium hydride (37.8 mmol) in 15 mL of dry dimethylformamide with stirring at 0 °C.…”

“…All CoA thioesters were detected at 260 nm and were further characterized by NMR or MALDI-TOF-MS analysis. Extinction coefficient values for the thioesters of DCTH-, CTFTH-, BTTB-, NPTB-, and DNPTB-CoA were used as reported previously (28). The extinction coefficients of the acid and coenzyme A thioester form of DCTFTH were reevaluated and determined to be 5.0 mM -1 cm -1 at 268 nm and 20.0 mM -1 cm -1 at 260 nm, respectively.…”

Acyl‐CoA dehydrogenases

“…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.…”

“…Baker‐Malcolm and C. Thorpe, unpublished data) that the catalytic base, Glu376, in MCAD is the target of this unusual reaction. The remaining four turnovers release the corresponding trans ‐2‐enoyl‐CoA product, which then serves as a substrate of enoyl‐CoA hydratase [102,105,106]. Consequently, the same thiolate species is released into the mitochondrial matrix from the thiohemiacetal product of the hydratase.…”