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 Escherichia coli were extended by replacement with Glu and/or Arg or by site-specific derivatization of single-Cys replacement mutants. Iodoacetic acid was used to carboxymethylate Cys, or methanethiosulfonate derivatives [Akabas, M. H., Stauffer, D. A., Xu, M., & Karlin, A. (1992) Science 258, 307] were used to attach negatively charged ethylsulfonate or positively charged ethylammonium groups. Replacement of Asp237 with Glu, carboxymethyl-Cys, or sulfonylethylthio-Cys yields active permease with Lys or Arg at position 358. Similarly, the permease tolerates 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 activity [Sahin-Tóth, M., Dunten, R. L., Gonzalez, A., & Kaback, H. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10547]. In contrast, replacement of Asp240 with Glu abolishes lactose transport, and permease with carboxymethyl-Cys, at position 240 is inactive when paired with Lys319, but it exhibits significant activity with Arg319. Interestingly, sulfonylethylthio-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 counterion, but no lactose transport is observed when either of these modifications is paired with Glu240.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Physiology and Microbiology, Howard Hughes Medical Institute, University of California at Los Angeles 90024-1570.
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D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008239:Lysine; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D001224:Aspartic Acid; C021499:lactose permease; D007785:Lactose; D008239:Lysine