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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
Author(s) -
Kimura Tomomi,
Inagaki Youko,
Sawai Tetsuo,
Yamaguchi Akihito
Publication year - 1995
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(95)00205-n
Subject(s) - antiporter , n ethylmaleimide , chemistry , mutant , substrate (aquarium) , stereochemistry , biochemistry , biology , gene , membrane , ecology
We previously reported that the reaction of [ 14 C] 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 [ 14 C]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.