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 loop2–3 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.
