The Trapping of Different Conformations of the Escherichia coli F1 ATPase by Disulfide Bond Formation
The Trapping of Different Conformations of the Escherichia coli F1 ATPase by Disulfide Bond Formation
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通过二硫键形成捕获大肠杆菌 F1 ATP 酶的不同构象
DOI：
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发表时间：
1996
影响因子：
4.8
通讯作者：
R. Capaldi
中科院分区：
文献类型：
--
作者：
G. Grüber;R. Capaldi
利用EscherichiacoliF 1ATP酶的两个突变体β Y331 W：E381 C/ε S108 C和α S411 C/β Y331 W/ε S108 C，研究了α3β3亚基结构域上茎形成亚基γ和ε的不同相互作用与催化位点上核苷酸结合的关系。在这些突变体中，通过Cu 2+诱导的二硫键形成，获得了β + γ和β + ε之间或α + γ和α + ε之间的基本上完全的产率交联，从而将酶捕获在与β或α亚基相互作用的小亚基的状态中。两种突变体中β Tyr-331的Trp的存在允许直接测量催化位点的核苷酸占用率。交联前，Mg 2 +ATP可结合在两种突变体的所有三个催化位点上，最高亲和力位点的Kd约为0.1 μM，第二和第三位点的Kd值分别约为2 μM和30-40 μM。在不存在Mg 2+的情况下，ATP也结合在所有三个催化位点上，但在两种突变体中具有单一的低亲和力（高于100 μM）。Cu 2+诱导的β Y331 W：E381 C/ε S108 C突变体ECF 1的交联对核苷酸结合的影响很小。Mg 2 +ATP的三个催化位点的结合亲和力没有显着改变交联前获得的，和酶之间的合作结合和平等的结合亲和力的三个催化位点（当Mg 2+是不存在的）仍然切换。当γ和ε亚基与α亚基交联时，ATP在最高亲和力催化位点的结合发生了显着改变。该位点变得封闭，使得在交联之前已经结合到其中的核苷酸（ATP或ADP）被捕获并且不能交换出来。此外，一旦酶交联，ATP或ADP不能进入该位点，尽管是空的。最后，γ和ε与α亚基的交联阻止了合作结合与三个催化位点相等的状态之间的转换。我们认为，小亚基位于α亚基的酶的构象发生在酶的功能过程中，在α3β3六聚体内的γ和ε亚基的旋转过程中，这可能是ATP合成的活化状态。
Two mutants of the Escherichia coli F1 ATPase, βY331W:E381C/εS108C and αS411C/βY331W/εS108C, have been used to relate nucleotide binding in catalytic sites with different interactions of the stalk-forming subunits γ and ε at the α3β3 subunit domain. Essentially full yield cross-linking between β + γ and β + ε, or between α + γ and α + ε, was obtained in these mutants by Cu2+-induced disulfide bond formation, thereby trapping the enzyme in states with the small subunits interacting either with β or α subunits. The presence of the Trp for β Tyr-331 in both mutants allowed direct measurement of nucleotide occupancy of catalytic sites. Before cross-linking, Mg2+ATP could be bound in all three catalytic sites in both mutants with a Kd of around 0.1 μM for the highest affinity site and Kd values of approximately 2 μM and 30-40 μM for the second and third sites, respectively. In the absence of Mg2+, ATP also bound in all three catalytic sites but with a single low affinity (above 100 μM) in both mutants. Cu2+-induced cross-linking of ECF1 from the mutant βY331W:E381C/εS108C had very little effect on nucleotide binding. The binding affinities of the three catalytic sites for Mg2+ATP were not significantly altered from those obtained before cross-linking, and the enzyme still switched between cooperative binding and equal binding affinities of the three catalytic sites (when Mg2+ was absent). When the γ and ε subunits were cross-linked to α subunits, ATP binding in the highest affinity catalytic site was dramatically altered. This site became closed so that nucleotide (ATP or ADP) that had been bound into it prior to cross-linking was trapped and could not exchange out. Also, ATP or ADP could not enter this site, although empty, once the enzyme had been cross-linked. Finally, cross-linking of the γ and ε to the α subunits prevented the switching between cooperative binding and the state where the three catalytic sites are equivalent. We argue that the conformation of the enzyme in which the small subunits are at α subunits occurs during functioning of the enzyme in the course of the rotation of γ and ε subunits within the α3β3 hexamer and that this may be the activated state for ATP synthesis.
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DOI：
--
发表时间：
1990-11
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Y. Milgrom;L. Ehler;P. Boyer
通讯作者：
Y. Milgrom;L. Ehler;P. Boyer
DOI：
10.1016/s0021-9258(20)80703-0
发表时间：
1993-09
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. Weber;S. Wilke-Mounts;Rita S. F. Lee;E. Grell;A. E. Senior
通讯作者：
J. Weber;S. Wilke-Mounts;Rita S. F. Lee;E. Grell;A. E. Senior
通过突变 α R376C 引入大肠杆菌 F1-ATP 酶的 α 亚基中的半胱氨酸靠近 α-β 亚基界面并靠近非催化核苷酸结合位点。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Turina,P;Aggeler,R;Lee,RS;Senior,AE;Capaldi,RA
通讯作者：
Capaldi,RA
水溶性碳二亚胺1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺对大肠杆菌F1的腺苷三磷酸酶活性的抑制是由于β亚基中几个羧基的修饰。
影响因子：
2.9
作者：
Lötscher,HR;deJong,C;Capaldi,RA
通讯作者：
Capaldi,RA
大肠杆菌腺苷三磷酸酶中ε亚基构象的催化位点核苷酸和无机磷酸盐依赖性。
影响因子：
2.9
作者：
Mendel-Hartvig,J;Capaldi,RA
通讯作者：
Capaldi,RA