PROPERTIES OF INTERACTING ASPARTIC-ACID AND LYSINE RESIDUES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

The side chains of the interacting pair Asp237(helix VII)-Lys358(helix XI) or Asp240(helix VII)-Lys319(helix X) in the lactose permease of E scherichia coli were extended by replacement with Glu and/or Arg or by site-specific derivatization of single-Cys replacement mutants. Iodoa cetic acid was used to carboxymethylate Cys, or methanethiosulfonate d erivatives [Akabas, M. H., Stauffer, D. A., Xu, M., & Karlin, A. (1992 ) Science 258, 307] were used to attach negatively charged ethylsulfon ate or positively charged ethylammonium groups. Replacement of Asp237 with Glu, carboxymethyl-Cys, or sulfonylethylthio-Cys yields active pe rmease with Lys or Arg at position 358. Similarly, the permease tolera tes replacement of Lys358 with Arg or ammonioethylthio-Cys with Asp or Glu at position 237. Remarkably, moreover, permease with Lys, Arg, or ammonioethylthio-Cys in place of Asp237 is highly active when Lys358 is replaced with Asp or Glu, in agreement with the conclusion that the polarity of the charge interaction can be reversed without loss of ac tivity [Sahin-Toth, M., Dunten, R. L., Gonzalez, A., & Kaback, H. R. ( 1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10547]. In contrast, replaceme nt of Asp240 with Glu abolishes lactose transport, and permease with c arboxymethyl-Cys, at position 240 is inactive when paired with Lys319, but it exhibits significant activity with Arg319. Interestingly, sulf onylethylthio-Cys substitution for Asp240 also results in significant transport activity. Permease with Arg or ammonioethylthio-Cys in place of Lys319 exhibits high activity with Asp240 as the negative counteri on, but no lactose transport is observed when either of these modifica tions is paired with Glu240. Lactose permease mutants in the charge pa ir Asp237-Lys358 are inserted into the membrane at wild-type levels if the charge pair is maintained with either polarity, while disruption of the interacting pair often causes a marked decrease in the amount o f protein inserted into the membrane; mutations in Asp240-Lys319 do no t affect insertion of the permease. The results demonstrate that neith er Asp237, Lys358, nor the interaction between these residues is impor tant for permease activity and that this putative salt bridge probably plays a role in membrane insertion. In contrast, the interaction betw een Asp240 and Lys319 exhibits much more stringent properties, and the polarity of the interaction appears to be important for activity.