Molecular Approach to the Regulatory Mechanism of Chloroplast ATP Synthase

本文研究了嗜热细菌F_1的cf_1 - γ亚基、α亚基和β亚基形成的嵌合物，通过寡核苷酸定向诱变技术修饰不同γ亚基的二硫桥形成或还原后，其催化活性的变化。为此，产生了三个突变体γ亚基：gamma_<DELTA194-230>，其中Pro-194至Ile-230缺失37个氨基酸；gamma_<C199A>， Cys-199变为Ala； gamma_<DELTA200-204>， Asp-200至Lys-204缺失氨基酸。与含有野生型CF_1-gamma的复合物相比，由这些突变型CF_1-gamma亚基和来自嗜热细菌F_1的α和β亚基产生的所有嵌合亚基复合物都失去了对巯基试剂的敏感性。最适pH值（pH 8.5 ~ pH 9.0）和刺激atp酶活性的甲醇浓度不受这些突变的影响。这表明，引入更多的突变并没有改变嵌合物的atp酶活性的主要特征。然而，γ亚基和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γ <DELTA200-204>或γ <C199A>的嵌合物的atp酶活性被来自CF_1的重组epsilon亚基抑制，类似于含有还原野生型γ亚基的复合物，但重组epsilon亚基不抑制含有氧化形式γ亚基的复合物的atp酶活性。这表明epsilon亚基对γ亚基的亲和力取决于γ亚基的状态，或者epsilon亚基可能以不抑制活性的方式与氧化形式的γ亚基结合。含有γ < delta194 -230>的复合物的atp酶活性未被epsilon亚基有效抑制。这些结果表明，γ亚基上二硫键的形成或减少可能引起该区域的构象变化，从而直接影响该亚基与邻近的epsilon亚基的相互作用。少

We have studied the change of the catalytic activity of chimeric complexes which were formed by CF_1-gamma, and alpha and beta subunits of thermophilic bacterial F_1 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, gamma_<C199A>, 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 CF_1-gamma subunits and alpha and beta subunits from thermophilic bacterial F_1 lost the sensitivity against thiol reagents when compared to the complex containing wild type CF_1-gamma. The pH optimum (pH 8.5 to pH 9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction o … More f 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 which contained gamma_<DELTA200-204> or gamma_<C199A> was inhibited by the addition of recombinant epsilon subunit from CF_1 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, suggesting the affinity of the epsilon subunit to the gamma subunit is 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 which contains gamma_<DELTA194-230> was not efficiently inhibited by epsilon subunit. These results show that the formation or the reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region which directly affects the interaction of this subunit with the adjacent epsilon subunit. Less