Delineating electrogenic reactions during lactose/H+ symport.
Delineating electrogenic reactions during lactose/H+ symport.
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DOI：
10.1021/bi100492p
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发表时间：
2010-07-27
期刊：
影响因子：
2.9
通讯作者：
Fendler, Klaus
中科院分区：
文献类型：
--
作者：
Garcia-Celma, Juan J.;Ploch, Julian;Smirnova, Irina;Kaback, H. Ronald;Fendler, Klaus
利用具有更高时间分辨率的固载膜电生理学技术，研究了乳糖/H+复合体在乳糖穿透酶(Lacy)催化下的电化学反应。分析了重组成蛋白脂质体的纯化的Lacy产生的电荷转移速率在从5.2%到8.5%的pH范围内，这允许表征运输机制中的两个电化学反应：(I)由糖结合触发的弱电生反应，并且在H+结合部位的Glu325→Ala突变或者被酸性pH取消的条件下观察到H+转位(对于野生型Lacy，这个步骤的速率常数为∼200 S−1导致中间产物被建议为代表被封闭的状态)和(Ii)对应于在pH 8的总转移电荷的94%的主要电化学反应，该酶的最大反应速率为∼30 S−1，pK为7.5.这种部分反应被认为是花边细胞质侧在周转过程中限速的H+释放。这些发现与以前的电生理结果和生化−生物物理研究一起被包括在一个总体动力学机制中，该机制允许描绘反应途径中的电产生步骤。
Electrogenic reactions accompanying downhill lactose/H+ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H+ translocation is abolished either by acidic pH or by a Glu325 → Ala mutation in the H+ binding site (this step with a rate constant of ∼200 s−1 for wild-type LacY leads to an intermediate proposed to represent an “occluded” state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ∼30 s−1 and a pK of 7.5. This partial reaction is assigned to rate-limiting H+ release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical−biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.
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影响因子：
2.9
作者：
GARCIA, ML;VIITANEN, P;KABACK, HR
通讯作者：
KABACK, HR
影响因子：
2.9
作者：
HERZLINGER, D;VIITANEN, P;KABACK, HR
通讯作者：
KABACK, HR
影响因子：
11.4
作者：
Mirza, O;Guan, L;Kaback, HR
通讯作者：
Kaback, HR
影响因子：
3.6
作者：
Saier, MH
通讯作者：
Saier, MH
DOI：
10.1073/pnas.1004515107
发表时间：
2010-05-25
影响因子：
11.1
作者：
Nie, Yiling;Kaback, H. Ronald
通讯作者：
Kaback, H. Ronald