Substrate‐induced acceleration of N‐ethylmaleimide reaction with the Cys‐65 mutant of the transposon Tn 10‐encoded metal‐tetracycline/H+ antiporter depends on the interaction of Asp‐66 with the substrate

FEBS Letters

Yamaguchi, Akihito¹  Kimura, Tomomi¹  Sawai, Tetsuo¹  Inagaki, Youko¹ 

[1] Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan

关键词: Antiporter;    Tetracycline/H+ antiporter;    Site-directed mutagenesis;    Sulfhydryl reagent;   

DOI  :  10.1016/0014-5793(95)00205-N

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We previously reported that the reaction of [¹⁴C]N-ethylmaleimide (NEM) with the S65C mutant of the transposon Tn10-encoded metal-tetracycline/H⁺ antiporter (TetA(B)) is competitively inhibited by tetracycline [Yamaguchi, A. et al., FEBS Lett. 322 (1993) 201–204]. However, this observation has been revealed to be a mistake. The reaction of [¹⁴C]NEM with S65C TetA(B) was significantly and reproducibly accelerated by tetracycline, i.e. not inhibited. When Asp-66 was replaced by Ala, the reaction of NEM with the Cys-65 residue was no longer affected by tetracycline. In contrast, when Arg-70 was replaced by Ala, the acceleration of the reaction was unaltered. The tetracycline acceleration of the reaction to the Cys-65 residue was further stimulated with energization of the membrane on the addition of NADH. On the other hand, the tetracycline-induced acceleration was not observed in the absence of a divalent cation. These observations indicated that the Cys-65 locus is exposed to the medium according to the interaction of a divalent cationtetracycline chelation complex with Asp-66.

【 授权许可】

Unknown