title: Site-directed mutagenesis of Pro327 in the lac permease of Escherichia coli.
abstract: By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease. Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease. Finally, permease with Leu at position 327 is completely inactive. The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327. It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important.
authors.affiliation: Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
authors.forename: J S
authors.initials: JS
authors.lastname: Lolkema
authors.forename: I B
authors.initials: IB
authors.lastname: Püttner
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics*; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011487:Protein Conformation; D027981:Symporters
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose