Inactivation of general acyl-CoA dehydrogenase from pig kidney by 2-alkynoyl coenzyme A derivatives: initial aspects

Abstract

Pig kidney general acyl-CoA dehydrogenase is rapidly, stoichiometrically, and irreversibly inactivated by the acetylenic thio ester 2-octynoyl coenzyme A (2-octynoyl-CoA). The inhibitor binds initially to the dehydrogenase with a 10-nm red shift and increased resolution of the flavin chromophore, followed by the generation of a charge-transfer complex between some form of the bound inhibitor and oxidized flavin (lambda max 800 nm; epsilon app = 4.5 mM-1 cm-1; k1 = 1.07 min-1, at pH 7.6, 25 degrees C). The rate of formation of the long wavelength band is increased markedly with increasing pH (pKapp = 7.9). This intermediate then decays with release of about 0.6 mol of CoASH at pH 7.6, yielding a final form with a spectrum typical of bound oxidized flavin. Both irreversible inactivation and covalent modification of the protein occur prior to the decay of the long wavelength species. The modified dehydrogenase is not reduced on prolonged anaerobic incubation with the substrate octanoyl-CoA. The inactive enzyme is unusually resistant to dithionite reduction but may be readily photoreduced via the blue semiquinone to the dihydroflavin form. This reduced enzyme is rapidly reoxidized by electron-transferring flavoprotein, the physiological electron acceptor of the dehydrogenase. General acyl-CoA dehydrogenase is also inactivated by 2-pentynoyl- and 2-pentadecynoyl-CoA with formation of an 800-nm band of lower intensity and by propiolyl-CoA, phenylpropiolyl-CoA, and 2-octynoylpantetheine without the appearance of detectable intermediate species. These data are compared with the behavior of acyl-CoA dehydrogenases toward mechanism-based inactivators carrying an acetylene function at C-3, e.g., 3-butynoyl-CoA.

Medium-chain acyl coenzyme A dehydrogenase from pig kidney has intrinsic enoyl coenzyme A hydratase activity

“…First, 2-octynoyl-CoA is a potent irreversible active site directed inactivator of the medium-chain acyl-CoA dehydrogenase attacking the protein, not the flavin, moiety (Freund et al, 1985). The modified enzyme is not reduced by octanoyl-CoA and apparently can no longer bind acetoacetyl-coA, a competitive inhibitor of the native enzyme (Freund et al, 1985).…”

“…The modified enzyme is not reduced by octanoyl-CoA and apparently can no longer bind acetoacetyl-coA, a competitive inhibitor of the native enzyme (Freund et al, 1985). In Figure 1, aliquots of the dehydrogenase were treated with increasing concentrations of 2-octynoyl-CoA, and each sample was assayed for both dehydrogenase and crotonase activity.…”

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

“…The apoenzyme was prepared according to ref 35 and had a residual activity 1-2% that of the holoenzyme. To eliminate any residual activity, the apoenzyme was incubated for 30 min at 4°C with 5 equiv (compared with residual holoenzyme) 2-octynoyl-CoA (36). Excess inactivator was subsequently eliminated by ultrafiltration.…”

Avian 3‐hydroxy‐3‐methylglutaryl‐CoA lyase: Sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines

“…3-Alkynoyl derivatives in particular have been shown to inactivate pig heart thiolase (Holland et al, 1973), butyryl-CoA dehydrogenase (Fendrich & Abeles, 1982), and pig liver general acyl-CoA dehydrogenase (Frerman et al, 1980). 2-Octynoyl-CoA has also been demonstrated to act as a suicide substrate for general acyl-CoA dehydrogenase (Freund et al, 1985).…”

“…2-Butynoyl-CoA was synthesized by the mixed anhydride procedure of Bernert and Sprecher (1977) as modified by Freund et al (1985) in their synthesis of 2-octynoyl-CoA. Freshly redistilled triethylamine (150 pmol) and 150 pmol ethylchloroformate were added to 125 pmol of 2-butynoic acid in 1 .O mL benzene.…”