Yabuki M, Nagakura T, Moritani C, Kanazawa H
Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Japan.
Arch Biochem Biophys. 1997 Feb 1;338(1):104-10. doi: 10.1006/abbi.1996.9805.
Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase. Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H. Kanazawa et al. (1995) Arch. Biochem. Biophys. 317, 348-356). Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase. Here, we introduced site-directed mutations into these residues. Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity. Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane. Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity. Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity. Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system. According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix. These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly.
单克隆抗体α110识别大肠杆菌F1 - ATP酶α亚基中的Leu - 456。该抗体与α亚基结合或此残基突变为Pro会导致ATP酶活性增强，这表明该残基参与了此分子的催化机制（H. 金泽等人（1995年）《生物化学与生物物理学报》317卷，348 - 356页）。Leu - 456与Gly - 454和Tyr - 455是不同物种羧基末端75个氨基酸中仅有的保守残基，这表明这三个残基在ATP酶的催化中起重要作用。在此，我们对这些残基进行了定点突变。不仅L456P，而且G454L、Y455K、Y455L和L456N突变都导致ATP酶活性增强。令人惊讶的是，Y455V、L456H和L456S导致F1亚基在膜上组装缺陷。用野生型β和γ亚基与突变型α亚基（L4γ6P）重建αβγ复合体表现出增强的ATP酶活性。向重建的F1 - ATP酶中添加分别与δ和ε亚基功能相似的与谷胱甘肽S - 转移酶融合的δ或ε，并未导致其活性显著增强。通过酵母双杂交系统检测到L456P突变导致α和β亚基之间的相互作用减弱。根据推导的牛α亚基三维结构，Leu - 456、Gly - 454和Tyr - 455包含在一个小的α螺旋中。这些结果表明，这个α螺旋影响α亚基与β亚基的相互作用，但不影响与δ或ε亚基的相互作用，这可能对催化机制和F1组装很重要。
