Site-directed mutagenesis of cysteine-148 in the Lac permease of Escherichia coli: effect on transport, binding, and sulfhydryl inactivation

Abstract

By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue [Trumble, W.R., Viitanen, P.V., Sarkar, H.K., Poonian, M.S., and Kaback, H.R. (1984) Biochem. Biophys. Res. Commun. 119, 860]. Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E. coli T184. Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl .alpha.-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease. In contrast, the Vmax for the lactose transport in vesicles containing mutant permease is significantly decreased. Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl .alpha.-D-galactopyranoside with high affinity. Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity. Importantly, .beta.-galactosyl 1-thio-.beta.-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant. The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease. Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl .alpha.-D-galactopyranoside from wild-type permease, does not affect binding in the mutant. The findings indicate that Cys148 is not obligatory for lactose/proton symport and that there is (are) another (other) Cys residue(s) in the permease that may be essential for activity. The other Cys residue(s) is (are) less reactive than Cys148, is (are) not protected against alkylation by .beta.-galactosyl 1-thio-.beta.-D-galactopyranoside, and is (are) not involved in binding of substrate.