ROLE OF THE CHARGE PAIR ASPARTIC ACID-237-LYSINE-358 IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI
ROLE OF THE CHARGE PAIR ASPARTIC ACID-237-LYSINE-358 IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI
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DOI：
10.1021/bi00063a028
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发表时间：
1993-03-30
期刊：
影响因子：
2.9
通讯作者：
KABACK, HR
中科院分区：
文献类型：
--
作者：
DUNTEN, RL;SAHINTOTH, M;KABACK, HR
利用缺乏Cys残基的乳糖渗透酶突变体（C-less permease），将Asp237和Lys358替换为Cys或其他氨基酸，以寻求这两个残基形成电荷对的建议[King， s.c., Hansen, c.l., & Wilson， T.H. (1991) Biochim]。Biophys。[j].科学通报，2009,33(2):387 - 398。单独用Cys、Ala或赖氨酸代替Asp237或用Cys、Ala或赖氨酸代替Lys358实际上消除了活性乳糖运输。然而，同时用Cys和/或Ala替换这两个残基会产生高活性的渗透酶。因此，任何位置的中性氨基酸取代都是有害的，因为它们使相反的电荷不成对。此外，引人注目的是，当Asp237与Lys358交换时，观察到高活性。结果强烈表明，Asp237和Lys358相互作用形成盐桥，残基和盐桥本身对活性都不重要。免疫结果显示，缺乏盐桥的活性突变体的膜水平较低，这表明盐桥在渗透酶折叠或稳定性中起作用，并提出盐桥可能存在于折叠中间而不存在于成熟蛋白中的可能性。然而，值得注意的是，用Cys代替Asp237的突变体通过羧基甲基化恢复了完全的活性，从而在位置237上重新产生了一个负电荷。脉冲追踪分析和热失活研究表明，237和358位Cys双突变体的稳定性与C-less相当。因此，Asp237和Lys358之间的相互作用可能对渗透酶折叠很重要，并且在成熟蛋白中保持。最后，在237和/或358位含有Cys的渗透酶分子，亲脂性比疏脂性巯基试剂更容易失活，尽管这两种类型的化合物失活的程度相同。因此，电荷对可能位于靠近膜-水界面的膜双层内，而不是位于膜的中间。
Using a lactose permease mutant devoid of Cys residues (C-less permease), Asp237 and Lys358 were replaced with Cys or other amino acids to pursue the proposal that the two residues form a charge pair [King, S.C., Hansen, C.L., & Wilson, T.H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Individual replacement of Asp237 with Cys, Ala, or Lys or replacement of Lys358 with Cys, Ala, or Asp virtually abolishes active lactose transport. However, simultaneous replacement of both residues with Cys and/or Ala yields permease with high activity. Therefore, neutral amino acid substitutions at either position are detrimental only because they leave the opposing charge unpaired. Strikingly, moreover, when Asp237 is interchanged with Lys358, high activity is observed. The results indicate strongly that Asp237 and Lys358 interact to form a salt bridge and that neither residue nor the salt bridge per se is important for activity. Immunoblots reveal low membrane levels of the active mutants lacking the putative salt bridge, suggesting a role for the salt bridge in either permease folding or stability and raising the possibility that the salt bridge may exist in a folding intermediate but not in the mature protein. Remarkably, however, a mutant with Cys in place of Asp237 is restored to full activity by carboxymethylation which recreates a negative charge at position 237. Pulse-chase analysis and heat-inactivation studies indicate that the stability of the double mutant with Cys at positions 237 and 358 is comparable to C-less. Therefore, the interaction between Asp237 and Lys358 is likely to be important for permease folding and is maintained in the mature protein. Finally, permease molecules with Cys at position 237 and/or 358 are inactivated more readily by lipophilic than lipophobic sulfhydryl reagents, although both types of compounds inactivate to the same extent. Thus, the charge pair may be located within the membrane bilayer close to the membrane-water interface rather than in the middle of the membrane.