Probing the mechanism of a membrane transport protein with affinity inactivators
Probing the mechanism of a membrane transport protein with affinity inactivators
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DOI：
10.1074/jbc.m211355200
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发表时间：
2003-03-21
影响因子：
4.8
通讯作者：
Kaback, HR
中科院分区：
文献类型：
--
作者：
Guan, L;Sahin-Tóth, M;Kaback, HR
亲和失活剂对于探索催化机理是有用的。在这里，我们描述了甲乙磺酰基（MTS）半乳糖或葡萄糖衍生物的合成和性质，以及一种研究得很好的膜转运蛋白，大肠杆菌的乳糖渗透酶。mts -半乳糖衍生物在缺乏Cys残基的背景下，作为具有Ala(122)- >Cys的功能性突变体的亲和灭活剂。质子电化学梯度（δ (H+)）显著提高了Cys（122）与mts -半乳糖衍生物之间的反应速率；相应的mts -葡萄糖衍生物的非特异性标记不受影响。当Ala(122)- >Cys突变与Cys(154)- >Gly突变结合时，mts -半乳糖和mts -葡萄糖衍生物之间的区别被消除，Delta（H+）没有影响。研究结果有力地证实，渗透酶底物的非半乳糖部分在螺旋IV中与Ala（122）相连。此外，研究结果还表明，mts -半乳糖衍生物本身并不与122位的Cys残基发生反应，而是在整个运输机制的后续步骤中发生反应。因此，这些灭活剂表现为独特的自杀底物。
Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala(122)-->Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (Delta(H+)) markedly increases the rate of reaction between Cys(122) and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala(122)-->Cys mutation is combined with a mutation (Cys(154)-->Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and MTS-glucose derivatives is abolished, and Delta(H+) has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala(122) in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.