Substrate-induced acceleration of N-ethylmaleimide reaction with the Cys-65 mutant of the transposon Tn10-encoded metal-tetracycline/H+ antiporter depends on the interaction of Asp-66 with the substrate.

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 cation-tetracycline chelation complex with Asp-66.