Schiele U, Niedermeier R, Stürzer M, Lynen F
Eur J Biochem. 1975 Dec 1;60(1):259-66. doi: 10.1111/j.1432-1033.1975.tb20998.x.
It was shown by gel electrophoresis in sodium dodecylsulphate solution that 3-methylcrotonyl-CoA carboxylase from Achromobacter IVS is composed of two different subunits with molecular weights of about 78000 and 96000, respectively. The biotin is bound to the heavier subunit. It was previously found that 3-methylcrotonyl-CoA carboxylase contains four biotin molecules per complex. A complex composed of four of each subunit would thus have a molecular weight of about 700000. This is compatible with the molecular weight of 760000 determined earlier by analytical ultracentrifugation. Both subunits were isolated preparatively. As the subunits, unlike the complex, are very sensitive to oxygen, special precautions had to be taken during isolation. The biotin-containing subunit was isolated by chromatography on DEAE-cellulose in 5 M urea. It no longer catalyzed the overall reaction, yet could still carboxylate free biotin. The biotin-free subunit was separated after dissociation of the enzyme by three-days' dialysis at pH 9.8 under nitrogen. On chromatography over a Sepharose-bound avidin column, the biotin-subunit was fixed and the biotin-free subunit was eluted unretarded. The latter subunit showed no enzymic activity. After the addition of the biotin-containing subunit, overall activity was regenerated. The speed of reassociation is very much enhanced by 3-methylcrotonyl-CoA. It was shown by reassociation experiments under different conditions that probably an initial complex, AxBy is formed, possessing a binding site for 3-methylcrotonyl-CoA. Upon the binding of this substrate the conformation may be changed to a form favourable for reconstitution. Finally, the structures of biotin enzymes from different sources are compared. In the course of evolution there is a tendency toward integration of the different constituent proteins into only one polypeptide chain.
在十二烷基硫酸钠溶液中进行的凝胶电泳表明，无色杆菌IVS的3-甲基巴豆酰辅酶A羧化酶由两种不同的亚基组成，其分子量分别约为78000和96000。生物素与较重的亚基结合。先前发现，3-甲基巴豆酰辅酶A羧化酶每个复合物含有四个生物素分子。因此，由每个亚基各四个组成的复合物的分子量约为700000。这与早期通过分析超速离心法测定的760000的分子量相符。两种亚基均通过制备方法分离。与复合物不同，亚基对氧气非常敏感，因此在分离过程中必须采取特殊预防措施。含生物素的亚基通过在5M尿素中于DEAE-纤维素上进行色谱分离。它不再催化整个反应，但仍能使游离生物素羧化。无生物素的亚基在氮气下于pH 9.8透析三天使酶解离后分离。在琼脂糖结合抗生物素蛋白柱上进行色谱分离时，含生物素的亚基被固定，无生物素的亚基未被阻滞而被洗脱。后一个亚基没有酶活性。加入含生物素的亚基后，恢复了整体活性。3-甲基巴豆酰辅酶A大大提高了重新结合的速度。在不同条件下进行的重新结合实验表明，可能形成了一种初始复合物AxBy，它具有一个3-甲基巴豆酰辅酶A的结合位点。该底物结合后，构象可能会改变为有利于重组的形式。最后，比较了不同来源的生物素酶的结构。在进化过程中，有将不同组成蛋白整合到仅一条多肽链中的趋势。
