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.
Kimura
T
T
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Inagaki
Y
Y
Sawai
T
T
Yamaguchi
A
A
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology; D011487:Protein Conformation; D013752:Tetracycline / Q000494:pharmacology*
D017920:Antiporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C079051:tetA protein, Bacteria; D009243:NAD; D001224:Aspartic Acid; D013752:Tetracycline; D008274:Magnesium; D003545:Cysteine; D005033:Ethylmaleimide