Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied.
Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied.
复制标题
DOI：
10.1016/s0021-9258(20)80703-0
复制
发表时间：
1993-09
期刊：
影响因子：
--
通讯作者：
J. Weber;S. Wilke-Mounts;Rita S. F. Lee;E. Grell;A. E. Senior
中科院分区：
文献类型：
--
作者：
J. Weber;S. Wilke-Mounts;Rita S. F. Lee;E. Grell;A. E. Senior
大肠杆菌F1-ATPase的残基βY331是从先前的亲和标记、诱变和LIN-苯并-ADP结合实验中得知的，它直接与催化位点结合的底物的腺嘌呤部分相互作用。在这里，我们将βY331突变为色氨酸。突变体细胞在琥珀酸或限制葡萄糖上生长良好，纯化的突变体F1具有与野生型相似的kappa、cat/kM和Lin-enzo-ADP结合特性。来自βW331残基的荧光在349 nm处显示最大值，表明空位处于极地环境。ATP、ADP或AMPPNP使βW331的荧光几乎完全猝灭，从而使占据催化位点的突变体F1的荧光与野生型相似。因此，βW331荧光提供了在真实平衡条件下核苷酸与催化位点结合的直接探针。我们在平行实验中测量了ATP的结合和水解，发现每个F1分子占据一个或两个催化位点并不能产生显着的水解率，而占据所有三个催化位则产生Vmax速率。Km(ATP)与Kd3相似，Kd3是ATP与第三催化位结合的Kd。我们还测定了AMPPNP和ADP的结合参数。对于二磷酸腺苷，第一催化位的“开”速率比以前的离心柱程序快得多(=5×10(5)M~(-1)S~(-1))，尽管Kd没有太大变化。AMPPNP第一部位的“ON”率比ADP或ATP低2个数量级，KD与ADP相似。
Residue beta Y331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and lin-benzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites. Here we mutagenized beta Y331 to tryptophan. Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kappa cat/Km and lin-benzo-ADP binding characteristics similar to wild type. Fluorescence from beta W331 residues exhibited a maximum at 349 nm, indicating a polar environment in unoccupied sites. ATP, ADP, or AMPPNP caused virtually complete quenching of beta W331 fluorescence, so that the fluorescence of mutant F1 with occupied catalytic sites resembled that of wild-type enzyme. Therefore the beta W331 fluorescence provided a direct probe of nucleotide binding to catalytic sites under true equilibrium conditions. We measured ATP binding and hydrolysis in parallel experiments and found that occupancy of one or two catalytic sites per F1 molecule did not yield significant rates of hydrolysis while occupancy of all three sites yielded Vmax rates. Km(ATP) was similar to Kd3, the Kd for ATP binding to the third catalytic site. We also measured AMPPNP and ADP binding parameters. For ADP, the “on” rate at the first catalytic site was much faster (> or = 5 x 10(5) M-1 s-1) than seen previously by centrifuge column procedures, although the Kd was not much changed. For AMPPNP, the “on” rate at the first site was 2 orders of magnitude less than for ADP or ATP, and the Kd was similar to that for ADP.