Matsui T, Muneyuki E, Honda M, Allison W S, Dou C, Yoshida M
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan.
J Biol Chem. 1997 Mar 28;272(13):8215-21. doi: 10.1074/jbc.272.13.8215.
A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.
来自嗜热芽孢杆菌PS3的F1 - ATP酶的突变α3β3γ复合物被构建出来，其中非催化性核苷酸结合位点失去了结合核苷酸的能力。它以协同动力学（Km(1)，4 μM；Km(2)，135 μM）的初始速率水解ATP，这与野生型复合物相似。然而，初始速率迅速衰减至失活形式。由于失活的突变复合物每摩尔复合物含有1.5摩尔ADP，这种失活似乎是由于在催化位点截留了抑制性的MgADP所致。事实上，突变复合物在与等摩尔MgADP预孵育10分钟后几乎完全失活。分析ATP水解开始后失活进程与ATP浓度的关系表明，在Km(1)范围内的ATP浓度下失活最为明显。在ATP存在的情况下，野生型复合物能解离预先加载到催化位点上的抑制性[3H]ADP，而突变复合物则不能。月桂基二甲基氧化胺以ATP依赖的方式促进预先加载的抑制性[3H]ADP的释放，并部分恢复失活突变复合物的活性。添加ATP促进了预先加载在突变复合物单个催化位点上的2',3'-O-(2,4,6-三硝基苯基)-ATP的单位点水解。这些结果表明，完整的非催化位点对于F1 - ATP酶的连续催化周转至关重要，但对于在ATP浓度低于约300 μM时观察到的F1 - ATP酶的催化协同性并非必不可少。
