Catalytic site nucleotide binding and hydrolysis in F1F0-ATP synthase.
Catalytic site nucleotide binding and hydrolysis in F1F0-ATP synthase.
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F1F0-ATP 合酶中的催化位点核苷酸结合和水解。
DOI：
10.1021/bi9807153
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Senior,AE
中科院分区：
文献类型：
--
作者：
Lobau,S;Weber,J;Senior,AE
F1Fo-ATP合成酶是从大肠杆菌β y331w突变体中纯化的。β-Trp-331提供了催化位点核苷酸结合的特异性荧光探针。生理浓度的底物MgATP填充了所有三个催化位点。mgtp和MgADP的催化位点表现出明显的协同性和不对称性，这取决于Mg2+。核苷酸结合速度快，kon= ~ 6 × 105M-1s-1。生理浓度（5mm）不与催化位点结合。在相同条件下测量MgATP的水解和结合，作为MgATP浓度的函数，表明只有在每个酶分子中填充所有三个催化位点时才能实现vmax。两个催化位点被占据而一个位点空的酶显示出低的非生理性催化速率。这是F1Fo中核苷酸结合参数的首次表征。纯化f1fo的行为在大多数方面与分离的f1相似，这一事实表明，额外的fo亚基sa、b和c的存在，以及固定的化学计量量的ε和δ的存在，并不影响催化位点的性质。结果影响了可能的催化机制，即，他们强调piccan不能简单地自发结合，具有所有三个位点的酶物种是唯一具有催化能力的物种，产物的释放和底物的结合不能同时发生，而前者必须在后者之前。
F1Fo-ATP synthase was purified fromEscherichia coliβY331W mutant. The β-Trp-331 provided a specific fluorescent probe of catalytic site nucleotide binding. Physiological (mM) concentration of substrate MgATP filled all three catalytic sites. With MgATP or MgADP the catalytic sites showed marked binding cooperativity and asymmetry, which was dependent on Mg2+. Nucleotide binding was fast, withkon= ∼6 × 105M-1s-1. Piat physiological concentration (5 mM) did not bind to catalytic sites. Measurement of MgATP hydrolysis and binding under identical conditions as a function of MgATP concentration revealed thatVmaxwas achieved only when all three catalytic sites were filled in every enzyme molecule. The enzyme species with two catalytic sites occupied and one site empty displayed low, nonphysiological catalytic rate. This is the first characterization of nucleotide binding parameters in F1Fo. The fact that the behavior of purified F1Fowas similar in most respects to that of isolated F1demonstrated that the presence of the additional Fosubunitsa,b, andc, and also fixed stoichiometric amounts of ε and δ, does not affect catalytic site properties. The results impact on possible catalytic mechanisms, namely, they emphasize that Picannot simply bind spontaneously, that an enzyme species with all three sites occupied is the only catalytically competent species, and that release of product and binding of substrate cannot be simultaneous, rather the former must precede the latter.