Hisabori T, Motohashi K, Kroth P, Strotmann H, Amano T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.
J Biol Chem. 1998 Jun 26;273(26):15901-5. doi: 10.1074/jbc.273.26.15901.
We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.
我们研究了由叶绿体偶联因子1（CF1）的γ亚基、嗜热细菌F1的α和β亚基形成的嵌合复合物在通过寡核苷酸定向诱变技术对不同γ亚基的二硫键进行形成或还原后催化活性的变化。为此，制备了三种突变γ亚基：γΔ194 - 230，其中从Pro - 194到Ile - 230的37个氨基酸被删除；γC199A，Cys - 199被改变为Ala；γΔ2,00 - 204，从Asp - 200到Lys - 204的氨基酸被删除。与含有野生型CF1 - γ的复合物相比，由这些突变CF1 - γ亚基中的每一个与嗜热细菌F1的α和β亚基产生的所有嵌合亚基复合物都失去了对硫醇试剂的敏感性。这些突变不影响最适pH（pH 8.5 - 9.0）和刺激ATP酶活性所需的甲醇浓度。这些表明突变的引入并没有改变嵌合复合物ATP酶活性的主要特征。然而，γ亚基与ε亚基之间的相互作用受到γ亚基本身类型的强烈影响。虽然含有γΔ2,00 - 204或γC199A的嵌合复合物的ATP酶活性与含有还原型野生型γ亚基的复合物类似，会被添加来自CF1的重组ε亚基所抑制，但重组ε亚基不会抑制含有氧化型γ亚基的复合物的ATP酶活性。因此，ε亚基对γ亚基的亲和力可能取决于γ亚基的状态，或者ε亚基可能以不抑制活性的方式与γ亚基的氧化形式结合。含有γΔ194 - 230的复合物的ATP酶活性没有被ε亚基有效抑制。这些结果表明，γ亚基上二硫键的形成或还原可能会在直接影响该亚基与相邻ε亚基相互作用的区域诱导构象变化。
