Engel P C, Jones J B
Biochem J. 1978 Apr 1;171(1):51-9. doi: 10.1042/bj1710051.
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.
醋酸苯汞可使埃氏巨球型菌的丁酰辅酶A脱氢酶可逆地褪色，消除710nm处的吸收带。鉴于以下新观察结果，重新审视了这是蛋白质巯基修饰结果的观点。(i) 用醋酸苯汞处理后，通过添加硫醇使酶重新变绿的能力不会因凝胶过滤或用硫酸铵沉淀而降低。(ii) 无论大量添加还是少量多次添加醋酸苯汞，其导致的褪色程度相同。然而，当少量多次添加时，褪色所需的总时间会大大延长。(iii) 在pH7.5的Tris/醋酸盐缓冲液中，完全褪色需要每摩尔酶亚基3.5摩尔醋酸苯汞，而在pH7的磷酸盐缓冲液中仅需2.5摩尔/摩尔。(iv) 与醋酸苯汞反应的基团均不能被碘乙酸或碘乙酰胺接触到。(v) 以摩尔计，二硫苏糖醇、巯基乙醇和辅酶A在使酶重新变绿方面同样有效。(vi) 只要醋酸苯汞不过量，褪色的酶能与巴豆酰辅酶A和乙酰乙酰辅酶A形成正常且稳定的复合物。(vii) 当存在少量过量的醋酸苯汞时，酶 - 乙酰乙酰辅酶A复合物的完全稳定形成需要每摩尔酶亚基添加几摩尔乙酰乙酰辅酶A。(viii) 褪色的酶被过量硫醇立即重新变绿的能力会随时间下降，在pH6时比在pH7或8时下降得更快，但在所有三个pH值下，瞬时重新变绿之后都会有一个A(710)进一步缓慢增加的阶段。在pH8时，这种进一步的恢复最为广泛和迅速。这些发现让人想起先前描述的绿色丁酰辅酶A脱氢酶的无蛋白酸性提取物重新变绿能力的可逆下降。得出的结论是，褪色的可能原因是紧密结合的硫酯的化学修饰，而非蛋白质巯基。如果硫酯存在于酶表面并与游离辅酶A和内酯处于平衡状态，或者如果酰基易于且可逆地从辅酶A的硫醇转移到蛋白质侧链，则可以解释这种可逆性。
