A reappraisal of the reaction of butyryl-coenzyme A dehydrogenase with phenylmercuric acetate. Evidence that de-greening involves a reaction of the tightly bound thioester.
Phenylmercuric acetate reversibly de-greens butyryl-CoA dehydrogenase from Megasphaera elsdenii, abolishing the absorption band at 710nm. The view that this is a result of modification of a protein thiol group is re-examined in the light of the following new observations. (i) After treatment with phenylmercuric acetate, the enzyme's ability to be re-greened by addition of thiols was not decreased by gel filtration or precipitation with (NH(4))(2)SO(4). (ii) Phenylmercuric acetate caused the same extent of de-greening whether added in a few large amounts or many small ones. The overall time taken for de-greening was, however, greatly extended when many small additions were made. (iii) In Tris/acetate buffer, pH7.5, 3.5mol of phenylmercuric acetate/mol of enzyme subunit was required for complete de-greening, compared with only 2.5mol/mol in phosphate buffer, pH7. (iv) None of the groups that react with phenylmercuric acetate is accessible to iodoacetate or iodoacetamide. (v) On a molar basis dithiothreitol, mercaptoethanol and CoA are equally effective in re-greening the enzyme. (vi) Provided that phenylmercuric acetate is not present in excess, the de-greened enzyme forms normal and stable complexes with crotonyl-CoA and acetoacetyl-CoA. (vii) When a small excess of phenylmercuric acetate is present, full stable development of the enzyme-acetoacetyl-CoA complex requires addition of several mol of acetoacetyl-CoA/mol of enzyme subunit. (viii) The ability of de-greened enzyme to be immediately re-greened by an excess of thiol declines with time, more rapidly at pH6 than at pH7 or 8, but at all three pH values the instantaneous re-greening was followed by a slow phase of further increase in A(710). This further recovery was most extensive and most rapid at pH8. These findings are reminiscent of the previously described reversible decline in the re-greening capacity of a protein-free acid extract of green butyryl-CoA dehydrogenase. It is concluded that the likely cause of de-greening is chemical modification of the tightly bound thioester rather than a protein thiol group. The reversibility would be explained if the thioester exists on the surface of the enzyme in equilibrium with free CoA and a lactone, or if the acyl group is readily and reversibly transferred from the thiol of CoA to a protein side chain.
A reappraisal of the reaction of butyryl-coenzyme A dehydrogenase with phenylmercuric acetate. Evidence that de-greening involves a reaction of the tightly bound thioester. Phenylmercuric acetate reversibly de-greens butyryl-CoA dehydrogenase from Megasphaera elsdenii, abolishing the absorption band at 710nm. The view that this is a result of modification of a protein thiol group is re-examined in the light of the following new observations. (i) After treatment with phenylmercuric acetate, the enzyme's ability to be re-greened by addition of thiols was not decreased by gel filtration or precipitation with (NH(4))(2)SO(4). (ii) Phenylmercuric acetate caused the same extent of de-greening whether added in a few large amounts or many small ones. The overall time taken for de-greening was, however, greatly extended when many small additions were made. (iii) In Tris/acetate buffer, pH7.5, 3.5mol of phenylmercuric acetate/mol of enzyme subunit was required for complete de-greening, compared with only 2.5mol/mol in phosphate buffer, pH7. (iv) None of the groups that react with phenylmercuric acetate is accessible to iodoacetate or iodoacetamide. (v) On a molar basis dithiothreitol, mercaptoethanol and CoA are equally effective in re-greening the enzyme. (vi) Provided that phenylmercuric acetate is not present in excess, the de-greened enzyme forms normal and stable complexes with crotonyl-CoA and acetoacetyl-CoA. (vii) When a small excess of phenylmercuric acetate is present, full stable development of the enzyme-acetoacetyl-CoA complex requires addition of several mol of acetoacetyl-CoA/mol of enzyme subunit. (viii) The ability of de-greened enzyme to be immediately re-greened by an excess of thiol declines with time, more rapidly at pH6 than at pH7 or 8, but at all three pH values the instantaneous re-greening was followed by a slow phase of further increase in A(710). This further recovery was most extensive and most rapid at pH8. These findings are reminiscent of the previously described reversible decline in the re-greening capacity of a protein-free acid extract of green butyryl-CoA dehydrogenase. It is concluded that the likely cause of de-greening is chemical modification of the tightly bound thioester rather than a protein thiol group. The reversibility would be explained if the thioester exists on the surface of the enzyme in equilibrium with free CoA and a lactone, or if the acyl group is readily and reversibly transferred from the thiol of CoA to a protein side chain.