Weber J, Senior A E
Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642, USA.
J Biol Chem. 1996 Feb 16;271(7):3474-7. doi: 10.1074/jbc.271.7.3474.
It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.
此前有人提出，F1 - ATP酶对2', 3'-O-(2,4,6-三硝基苯基)腺苷5'-三磷酸（TNP - ATP）的Vmax水解速率仅需该酶上两个催化位点被占据（格鲁布迈尔，C.，和佩内夫斯基，H. S.（1981年）《生物化学杂志》256卷，3718 - 3727页），然而最近有研究表明，ATP水解的Vmax速率要求所有三个催化位点都被占据（韦伯，J.，威尔克 - 芒茨，S.，李，R. S. F.，格雷尔，E.，和西尼尔，A. E.（1993年）《生物化学杂志》268卷，20126 - 20133页）。为了解决这一明显的差异，我们在相同条件下，使用βY331W突变型大肠杆菌F1 - ATP酶测量了MgTNP - ATP的平衡结合与水解情况，其中基因工程改造的色氨酸可对催化位点的占据情况提供直接的荧光探针。我们发现，MgTNP - ATP以Vmax速率水解确实需要所有三个催化位点都被占据，但与MgATP的情况不同，MgTNP - ATP的“双位点水解”占Vmax的很大一部分（约40%）。MgTNP - ATP与三个催化位点的结合表现出很强的结合协同性（Kd1 < 1纳米，Kd2 = 23纳米，Kd3 = 1.4微摩尔）。游离的TNP - ATP（即在存在EDTA的情况下）与所有三个催化位点结合的亲和力较低且不被水解。这些数据强调，Mg2 + 的存在对于F1 - ATP酶中底物结合的协同性、极高亲和力的第一个催化位点的形成以及水解活性至关重要，并且这三个特性密切相关。
