Hu D, Fiedler H R, Golan T, Edelman M, Strotmann H, Shavit N, Leu S
Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
J Biol Chem. 1997 Feb 28;272(9):5457-63. doi: 10.1074/jbc.272.9.5457.
The participation of the amino acid beta83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid beta83 was changed from Glu to Asp (betaE83D) and to Lys (betaE83K), and the highly conserved tetrapeptide betaT82-E83-G84-L85 (DeltaTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15DeltaatpB and FUD50 with the mutated atpB genes. The transformants containing the betaE83D and betaE83K mutant genes grew well photoautotrophically. The DeltaTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped DeltapH with thylakoids isolated from cw15 and the two mutants, betaE83D and betaE83K, were similar. However, only the phosphorylation activity of the mutant betaE83D was inhibited by tentoxin with 50% inhibition attained at 4 microM. These results confirm that amino acid beta83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, betaE83D, and betaE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the betaE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the betaE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the betaE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF1-ATPase.
先前有报道称，氨基酸β83参与决定叶绿体ATP合酶对抗霉素的敏感性。我们通过定点诱变改变了莱茵衣藻atpB基因的第83位密码子，以进一步研究该氨基酸在ATP合酶对抗霉素的反应以及ATP合成与水解机制中的作用。将氨基酸β83由Glu分别变为Asp（βE83D）和Lys（βE83K），并删除了高度保守的四肽βT82 - E83 - G84 - L85（ΔTEGL）。通过用突变的atpB基因对atpB缺失突变体cw15ΔatpB和FUD50进行粒子枪转化，获得了突变菌株。含有βE83D和βE83K突变基因的转化体在光自养条件下生长良好。ΔTEGL转化体不能进行光自养生长，使用CF1特异性抗体进行蛋白质免疫印迹染色时，未检测到CF1亚基。从cw15以及两个突变体βE83D和βE83K分离得到的类囊体在钳制的ΔpH下的ATP合成速率相似。然而，只有突变体βE83D的磷酸化活性受到抗霉素的抑制，在4μM时抑制率达到50%。这些结果证实，氨基酸β83在决定ATP合酶对抗霉素的反应中起关键作用。从cw15、βE83D和βE83K分离得到的CF1的潜在Mg - ATP酶活性速率相似，并且可以通过加热、醇类和辛基葡糖苷增强。与膜结合酶的情况一样，只有来自βE83D突变体的CF1对抗霉素敏感。与其他两个菌株的CF1相比，βE83K - CF1的ATP酶最佳刺激所需的醇浓度更低。此外，βE83K - CF1的最佳活性也更低。这些结果表明，在“冠”结构域的第83位引入带正电荷侧链的氨基酸会影响CF1 - ATP酶的活性构象。
