Effects of sulfhydryl reagents on the Cys65 mutant of the transposon Tn10‐encoded metal‐tetracycline/H+ antiporter of Escherichia coli

FEBS Letters

Yamaguchi, Akihito¹  Kimura, Tomomi¹  Sawai, Tetsuo¹ 

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

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

DOI  :  10.1016/0014-5793(93)81568-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The Cys⁶⁵ mutant of the Tn10-encoded metal-tetracycline/H⁺ antiporter is the only one which is inactivated by sulfhydryl reagents among the Cys mutants of the putative loop_2–3 region [Yamaguchi, A. et al. (1992) J. Biol. Chem. 267, 19155–19162]. The tetracycline transport activity of the Cys⁶⁵ mutant was completely abolished by N-ethylmaleimide; however, methyl methanethiosulfonate only abolished 45% of the activity, even in the presence of an excess of the reagent. Since N-ethylmaleimide did not further inactivate the methyl methanethiosulfonate-treated antiporter, it is clear that the modified antiporter molecule with a small substituent, a thiomethyl group, had significant but lower activity than the unmodified antiporter. The binding of [¹⁴C]N-ethylmaleimide to the Cys⁶⁵ mutant was inhibited in the predsence of tetracycline. These findings indicate that position 65 is close to the site of the interaction with the substrate and the modification of the side chain at this position caused steric hindrance as to substrate translocation.

【 授权许可】

Unknown