The α3(βMet222Ser/Tyr345Trp)3γ subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity
The α3(βMet222Ser/Tyr345Trp)3γ subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity
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TF1-ATPase 的 α3(βMet222Ser/Tyr345Trp)3γ 亚复合物在底物与最低亲和力的催化位点结合之前不会以显着的速率水解 ATP
DOI：
10.1021/bi060232w
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
W. Allison
中科院分区：
文献类型：
--
作者：
H. Ren;S. Bandyopadhyay;W. Allison
嗜热芽孢杆菌α3(βM222S/Y345W)的γ双突变亚复合体不含内源核苷酸，在翻转过程中不会将抑制作用的mADP包埋在催化部位。它对100nM−2mM三磷酸腺苷的水解度为31μM，kCAT为220kCAT-1。荧光滴定表明，这两种镁−核苷酸络合物均与亲和力最高的催化部位结合，Kd1值小于1 nM，与中等亲和力的催化部位结合，共同Kd2值约为12 nM。用二磷酸镁和三磷酸镁滴定得到亲和力最低的催化位的Kd3值分别为25和37μM。该双突变体对200 nM三磷酸腺苷的一级水解率为1.5kCAT-1，占kCAT的0.7%。因此，当中等亲和力的催化位饱和并且最低亲和力的催化位被最小限度地占据时，它不会以显著的速度水解ATP。在加入斯多葛之后。
The α3(βM222S/Y345W)3γ double-mutant subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 (TF1), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM−2 mM ATP with a Km of 31 μM and a kcat of 220 s-1. Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg−nucleotide complexes bind to the catalytic site of the highest affinity with Kd1 values of less than 1 nM and bind to the site of intermediate affinity with a common Kd2 value of about 12 nM. The Kd3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 μM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s-1, which is 0.7% of kcat. Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoic...