Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis.
Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis.
复制标题
乳糖和磷酸化磷酸化载体蛋白 HPr(Ser-P) 与短乳杆菌的乳糖/H 共向通透酶的协同结合。
DOI：
10.1073/pnas.92.2.417
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发表时间：
1995
影响因子：
11.1
通讯作者：
SaierJr,MH
中科院分区：
文献类型：
--
作者：
Ye,JJ;SaierJr,MH
短乳杆菌通过糖/H+同向转运积累乳糖和不可代谢的乳糖类似物，但向细胞外培养基中加入葡萄糖会导致游离糖从细胞中快速流出，这是由于糖转运与质子转运解偶联。利用L. brevis细胞，我们最近表明，这些调节/效果可以归因于代谢物激活的ATP依赖性蛋白激酶催化的磷酸转移酶系统的磷酸载体蛋白HPr [HPr（Ser-P）]中丝氨酸-46的磷酸化，并且具有丝氨酸-46->天冬氨酸替换的HPr突变形式（[S46 D]HPr）显然锁定在丝氨酸磷酸化构象中。我们在这里证明，[S46 D]HPr直接结合到由内而外的膜囊泡的L。含有乳糖通透酶的短杆菌。通透酶的糖底物显着和特异性地刺激[S46 D]HPr与膜的结合，而某些转运抑制剂如N-乙基马来酰亚胺阻断结合。结合的pH依赖性遵循运输的pH依赖性。野生型HPr和[S46 A]HPr突变体蛋白没有明显竞争与[S46 D]HPr结合的通透酶。这些结果为HPr（Ser-P）与L.乳糖/质子同向转运体上的变构位点直接相互作用提供了证据。brevis的目的是响应细胞的代谢需要调节糖积累。
Lactobacillus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles of L. brevis cells, we recently showed that these regulatory/effects could be attributed to the metabolite-activated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46 in the phosphocarrier protein HPr [HPr(Ser-P)] of the phosphotransferase system and that a mutant form of HPr with the serine-46-->aspartate replacement ([S46D]HPr) is apparently locked in the seryl phosphorylated conformation. We here demonstrate that [S46D]HPr binds directly to inside-out membrane vesicles of L. brevis that contain the lactose permease. Sugar substrates of the permease markedly and specifically stimulate binding of [S46D]HPr to the membranes while certain transport inhibitors such as N-ethylmaleimide block binding. The pH dependency for binding follows that for transport. Wild-type HPr and the [S46A]HPr mutant protein did not appreciably compete with [S46D]HPr for binding to the permease. These results provide evidence for the direct interaction of HPr(Ser-P) with an allosteric site on the lactose/proton symporter of L. brevis for the purpose of regulating sugar accumulation in response to the metabolic needs of the cell.