Hara K Y, Kato-Yamada Y, Kikuchi Y, Hisabori T, Yoshida M
Chemical Resources Laboratory, R-1, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama, 226-8503, Japan.
J Biol Chem. 2001 Jun 29;276(26):23969-73. doi: 10.1074/jbc.M009303200. Epub 2001 Mar 28.
In F(1)-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260-14263), and the role of this motif remained unknown. Here, we found that the epsilon subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F(1)-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the epsilon subunit in the mutant F(1)-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the epsilon subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the beta subunit or of the basic residues in the C-terminal alpha-helix of the epsilon subunit induced a partial effect. We here conclude that the epsilon subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the beta subunit.
在F(1)-ATP酶（一种旋转马达酶）中，当核苷酸填充催化位点时，β亚基中保守的DELSEED基序区域会移动并与转子γ亚基接触，并且该基序的酸性性质先前被认为在旋转中起关键作用。然而，我们之前的工作推翻了这一假设（原，K.Y.，野地，H.，鲍尔德，D.，安田，R.，木下，K.，Jr.，和吉田，M.（2000年）《生物化学杂志》275，14260 - 14263），并且该基序的作用仍然未知。在这里，我们发现ε亚基（一种内在抑制剂）无法抑制突变嗜热F(1)-ATP酶的ATP酶活性，在该突变体中DELSEED基序中的所有五个酸性残基都被丙氨酸取代，尽管突变F(1)-ATP酶中的ε亚基呈现抑制形式。此外，用丙氨酸取代ε亚基C末端区域的碱性残基会导致抑制作用降低。部分取代β亚基DELSEED基序中的酸性残基或ε亚基C末端α螺旋中的碱性残基会产生部分影响。我们在此得出结论，ε亚基通过与β亚基的DELSEED基序的静电相互作用发挥其抑制作用。
