Changing the lactose permease of Escherichia coli into a galactose-specific symporter

摘要

N-ethylmaleimide (NEM) modification of a lactose permease mu- tant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and β,D- galactopyranosyl 1-thio-β,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [~14C]NEM. Remarkably, however, D-galactose transport is rela- tively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consis- tently, competitive inhibition of [~14C]galactose transport by lac- tose, melibiose, or β,D-galactopyranosyl 1-thio-β,D-galactopyrano- side is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected.