Dunten R L, Sahin-Tóth M, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1574.
Biochemistry. 1993 Mar 30;32(12):3139-45. doi: 10.1021/bi00063a028.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
利用一个不含半胱氨酸残基的乳糖通透酶突变体（无半胱氨酸通透酶），将天冬氨酸237和赖氨酸358替换为半胱氨酸或其他氨基酸，以探究这两个残基形成电荷对的假说[金，S.C.，汉森，C.L.，& 威尔逊，T.H.（1991年）《生物化学与生物物理学报》1062，177 - 186]。将天冬氨酸237分别替换为半胱氨酸、丙氨酸或赖氨酸，或者将赖氨酸358分别替换为半胱氨酸、丙氨酸或天冬氨酸，实际上都会消除乳糖的主动转运。然而，将这两个残基同时替换为半胱氨酸和/或丙氨酸会产生具有高活性的通透酶。因此，在任一位置进行中性氨基酸替换是有害的，仅仅是因为它们使相反电荷未配对。此外，引人注目的是，当天冬氨酸237与赖氨酸358互换时，会观察到高活性。结果有力地表明，天冬氨酸237和赖氨酸358相互作用形成盐桥，并且这两个残基本身以及盐桥本身对活性都不重要。免疫印迹显示，缺乏假定盐桥的活性突变体的膜水平较低，这表明盐桥在通透酶折叠或稳定性方面发挥作用，并增加了盐桥可能存在于折叠中间体而非成熟蛋白中的可能性。然而，值得注意的是，用半胱氨酸取代天冬氨酸237的突变体通过羧甲基化恢复了全部活性，羧甲基化在位置237重新产生了负电荷。脉冲追踪分析和热失活研究表明，在位置237和358带有半胱氨酸的双突变体的稳定性与无半胱氨酸通透酶相当。因此，天冬氨酸237和赖氨酸358之间的相互作用可能对通透酶折叠很重要，并在成熟蛋白中得以维持。（摘要截断于250字）
