Lessard I A, Perham R N
Department of Biochemistry, University of Cambridge, U.K.
Biochem J. 1995 Mar 15;306 ( Pt 3)(Pt 3):727-33. doi: 10.1042/bj3060727.
The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.
对嗜热脂肪芽孢杆菌丙酮酸脱氢酶多酶复合物中二氢硫辛酰乙酰基转移酶（E2）组分的双结构域（硫辛酰结构域加外周亚基结合结构域）与丙酮酸脱羧酶（E1）组分之间的相互作用进行了研究。每1摩尔异源四聚体E1（α2β2）仅结合1摩尔双结构域（结合结构域），且这种结合对E1的动力学活性没有影响。同样，双结构域以最大多肽链比例1:2与单独的E1β亚基结合，但未发现与E1α亚基有可检测到的相互作用。然而，将单体E1α亚基添加到E1β - 双结构域复合物中可生成功能完整的E1（α2β2） - 双结构域复合物，这表明E1β亚基在将E1组分与双结构域结合中起关键作用，也表明在体外促进三种独立蛋白质组装不需要伴侣蛋白。在双结构域存在的情况下混合E1和二氢硫辛酰脱氢酶（E3）组分，结果显示E1和E3不能同时结合到同一分子的双结构域上，这是组装途径的一个新特征，也是决定组装酶复合物最终结构的一个重要因素。
