Jault J M, Matsui T, Jault F M, Kaibara C, Muneyuki E, Yoshida M, Kagawa Y, Allison W S
Department of Chemistry and Biochemistry 0601, University of California at San Diego, La Jolla 92093-0601, USA.
Biochemistry. 1995 Dec 19;34(50):16412-8. doi: 10.1021/bi00050a023.
ATP hydrolyses by the wild-type alpha 3 beta 3 gamma and mutant (alpha D261N)3 beta 3 gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 microM ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate. In contrast, the mutant complex hydrolyzes 50 microM or 2 mM ATP in two kinetic phases. The mutation abolishes acceleration from the intermediate phase to a faster final rate. Both the wild-type and mutant complexes hydrolyze ATP with a lag after loading a catalytic site with MgADP. The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex. The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant apo-complex. Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively. The rate of release of [3H]ADP from the Mg[3H]ADP-loaded mutant complex during hydrolysis of 40 microM ATP is slower than observed with the wild-type complex. LDAO increases the rate of release of [3H]ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 microM ATP. Again, release is slower with the mutant complex. When the wild-type and mutant complexes are irradiated in the presence of 2-N3-[3H]ADP plus Mg2+ or 2-N3-[3H]ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed. Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N3-[3H]ADP or 2-N3-[3H[ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex. The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.
对嗜热芽孢杆菌PS3的F1 - ATP酶的野生型α3β3γ和突变型（αD261N）3β3γ亚复合物的ATP水解进行了比较。野生型复合物以三个动力学阶段水解50微摩尔ATP：一个爆发阶段减速至中间阶段，然后逐渐加速至最终速率。相比之下，突变型复合物以两个动力学阶段水解50微摩尔或2毫摩尔ATP。该突变消除了从中间阶段到更快最终速率的加速过程。野生型和突变型复合物在催化位点加载MgADP后都有滞后地水解ATP。加载MgADP的野生型复合物的速率迅速加速并接近野生型脱辅基复合物所观察到的速率。加载MgADP的突变型复合物水解ATP时具有更明显的滞后，并且逐渐加速的速率接近突变型脱辅基复合物所观察到的缓慢最终速率。月桂基二甲基氧化胺（LDAO）分别刺激野生型和突变型复合物催化的2毫摩尔ATP水解4倍和7.5倍。在40微摩尔ATP水解过程中，从加载Mg[3H]ADP的突变型复合物中释放[3H]ADP的速率比野生型复合物慢。LDAO增加了在40微摩尔ATP水解过程中从预加载的野生型和突变型复合物中释放[3H]ADP的速率。同样，突变型复合物的释放较慢。当野生型和突变型复合物在2 - N3 - [3H]ADP加Mg2 +或2 - N3 - [3H]ATP加Mg2 +和叠氮化物存在下进行辐照时，观察到非催化位点的标记程度相同。虽然ADP和ATP分别对野生型和突变型复合物的非催化位点提供大致相同程度的保护，使其免受2 - N3 - [3H]ADP或2 - N3 - [3H]ATP的标记，但AMP - PNP对突变型复合物的非催化位点几乎没有保护作用。结果表明，该取代并不阻止ADP或ATP与非催化位点的结合，而是影响配体化的非催化位点与催化位点之间的串扰，而这种串扰对于促进抑制性MgADP的解离是必需的。
