BACKGROUND & AIMS:
:Upon modification of the reconstituted aspartate/glutamate carrier by mercury reagents the antiporter was converted into a unidirectional efflux carrier (Dierks, T., Salentin, A., Heberger, C. and Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268). In addition to this basic change in the mechanism, the mercurials, reacting with exofacial cysteines, also affected the internal binding site of the carrier leading to an unmeasurable high Km and to a drastically reduced substrate specificity. The spectrum of efflux substrates comprised small anions from chloride to glutamate, but not cationic amino acids and ATP, hence resembling pore-like properties. However, in the efflux state important carrier properties were also observed. The activation energy (86 kJ/mol) was as high as for the antiport. Furthermore, efflux was inhibited by the presence of external substrate. This trans-inhibition strongly suggests that the external binding site of the carrier, prerequisite in the antiport mechanism, also is involved in conformational transitions during efflux function. However, antiport no longer is catalyzed after switching to the efflux state. Reversion of the induced efflux carrier to the antiport state was achieved using dithioerythritol, thereby further restoring substrate specificity and saturation kinetics. A model for antiport-efflux interconversion is presented suggesting that two reactive cysteines have to be modified in order to uncouple the inward and outward directed component of antiport. The pore-type characteristics of efflux are taken as evidence that a channel-like structure determines the selectivity of unidirectional transport. This intrinsic channel of the protein then is required for substrate translocation also during antiport function.
背景与目标:
:在通过汞试剂修饰重构的天冬氨酸/谷氨酸载体后,反转运蛋白转化为单向外排载体(Dierks,T.,Salentin,A.,Heberger,C. andKrämer,R.(1990)Biochim。Biophys。Acta 1028,268)。除了机理上的这一基本变化外,与远颌半胱氨酸反应的汞也影响了载体的内部结合位点,导致无法测量的高Km并大大降低了底物特异性。外排底物的光谱包含从氯离子到谷氨酸的小阴离子,但不包含阳离子氨基酸和ATP,因此具有类似孔的特性。但是,在外排状态下,也观察到重要的载流子性质。活化能(86 kJ / mol)和反端口一样高。此外,外基质的存在抑制了外排。这种反式抑制作用强烈暗示了载体的外部结合位点是反端口机制的先决条件,在外排功能过程中也参与构象转变。但是,切换到外排状态后不再催化反端口。使用二硫赤藓糖醇将诱导的外排载体还原为反端口状态,从而进一步恢复底物特异性和饱和动力学。提出了反端口-外向互转换的模型,该模型建议必须对两个反应性半胱氨酸进行修饰,以解开反端口的向内和向外定向成分。外排的孔型特征被认为是通道状结构决定了单向转运的选择性的证据。然后,在反转运功能期间,该蛋白质的该固有通道也是底物转运所必需的。