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The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur.

ABSTRACT: Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic sulphur is shown to involve chemical modification of the tightly bound CoA persulphide. 5,5'-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) de-greens the enzyme essentially irreversibly, with a stoicheiometry of approx. 1 mol/mol of FAD. A compound separated by subsequent gel filtration is eluted at the same position as a CoA-thionitrobenzoate standard. The 35S-labelled distal sulphur atom of CoA persulphide is separated from this material. The enzyme remains fully active. Phenylmercuric acetate also de-greens the enzyme. The extent to which thiols restore the green colour declines with time. Gel filtration of mercurial-treated enzyme separates low-Mr material containing a CoA moiety, an extra S atom and a phenylmercury moiety. This material, added to yellow butyryl-CoA dehydrogenase with an excess of thiol, re-forms green enzyme, but it loses this ability on storage. The results are explicable if it is assumed that the thionitrobenzoate derivative of CoA persulphide loses the extra sulphur atom much more readily than does the phenylmercury derivative.

SUBMITTER: Williamson G

PROVIDER: S-EPMC1154400 | biostudies-other | 1983 Jun

REPOSITORIES: biostudies-other

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Project description:The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.

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The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur.

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