Molecular Approach to the Regulatory Mechanism of Chloroplast ATP Synthase

我们研究了用寡核苷酸定向诱变技术修饰不同伽马亚基的二硫键形成或还原后，与嗜热细菌F_1的α、β亚基形成的嵌合复合物催化活性的变化。为此目的，产生了三个突变的伽马亚基：GAMA_&lt；DELTA194-230&gt；其中，从Pro-194到ILE-230的37个氨基酸被删除，GAMA_&lt；C199A&gt；，Cys-199被改变为Ala，而从Asp-200到Lys-204的氨基酸被删除。与含有野生型CF1-伽马亚基的复合体相比，所有这些突变的CF1-伽马亚基和嗜热细菌F1的α、β亚基产生的嵌合亚基复合体对硫醇试剂都失去了敏感性。这些突变不影响ATPase活性的最适pH(pH 8.5~9.0)和甲醇浓度。这些都表明…的引入更多的突变并没有改变嵌合复合体的ATPase活性的主要特征，但伽马亚基和epsilon亚基之间的相互作用强烈地受到伽马亚基类型本身的影响。虽然类似于含有还原野生型伽马亚基的复合物一样，通过添加来自CF1的重组epsilon亚基来抑制含有伽马亚基的嵌合复合体的ATPase活性，但重组的epsilon亚基并不抑制含有氧化形式的伽马亚基的络合物的ATPase，这表明epsilon亚单位与伽马亚单位的亲和力取决于伽马亚单位的状态，或者epsilon亚单位可能以不抑制活性的方式与氧化形式的伽马亚基结合。β亚基不能有效地抑制含有γ-lt；DELTA194-230的复合体的ATPase活性。这些结果表明，γ亚基上二硫键的形成或还原可能会引起该区域的构象变化，从而直接影响该亚基与相邻的epsilon亚基的相互作用。较少

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