Majumdar R, Guest J R, Bridger W A
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochim Biophys Acta. 1991 Jan 8;1076(1):86-90. doi: 10.1016/0167-4838(91)90223-m.
Succinyl-CoA synthetase (EC 6.2.1.5, succinate:CoA ligase (ADP-forming] of Escherichia coli is an alpha 2 beta 2 tetramer, with the active site believed to be located at the point of contact between the two subunit types. It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis. The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the alpha subunit, and the succinate and CoA binding sites are in beta. A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction. As expected, the His-246 alpha----Asp mutant is incapable of undergoing phosphorylation. We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the beta subunit; this reaction does not involve covalent participation of His-246 alpha. We have found that the His-246 alpha----Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 alpha is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.
琥珀酰辅酶A合成酶（大肠杆菌的EC 6.2.1.5，琥珀酸：辅酶A连接酶（形成ADP））是一种α2β2四聚体，其活性位点据信位于两种亚基类型的接触点。先前已经确定，该反应在催化过程中涉及磷酸化酶形式的中间参与。磷酸化位点（His-246）以及底物ADP和ATP的结合位点位于α亚基中，而琥珀酸和辅酶A的结合位点在β亚基中。已经大量产生了这种酶的一种突变形式，其中活性位点的组氨酸残基被天冬氨酸取代，并纯化至同质。这种形式在亚基组装方面似乎与天然酶没有区别，但没有催化整个反应的能力。正如预期的那样，His-246α→Asp突变体无法进行磷酸化。我们开发了一种基于琥珀酰辅酶A砷解作用的测定方法，该方法有效地分离了在β亚基贡献的活性位点部分中发生的部分反应；该反应不涉及His-246α的共价参与。我们发现His-246α→Asp突变体在这种砷解反应中也没有活性，这表明完整的His-246α对于在活性位点的这一部分中建立整体反应相应步骤所需的微环境是必需的。
