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Effects of sulfhydryl reagents on the Cys 65 mutant of the transposon Tn10‐encoded metal‐tetracycline/H + antiporter of Escherichia coli
Author(s) -
Yamaguchi Akihito,
Kimura Tomomi,
Sawai Tetsuo
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81568-k
Subject(s) - tn10 , escherichia coli , transposable element , antiporter , tetracycline , chemistry , mutant , dna transposable elements , transposase , microbiology and biotechnology , biology , antibiotics , biochemistry , gene , membrane
The Cys 65 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 Cys 65 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 [ 14 C] N ‐ethylmaleimide to the Cys 65 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.