title: Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding.
abstract: The lactose permease (LacY) catalyzes galactoside/H(+) symport via an alternating access mechanism in which sugar- and H(+)-binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by opening and closing of inward- and outward-facing cavities. The crystal structures of wild-type LacY, as well as accessibility data for the protein in the membrane, provide strong support for a conformation with a tightly closed periplasmic side and an open cytoplasmic side (an inward-facing conformation). In this study, rates of substrate binding were measured by stopped-flow with purified LacY either in detergent or in reconstituted proteoliposomes. Binding rates are compared with rates of sugar-induced opening of the periplasmic pathway obtained by using a recently developed method based on unquenching of Trp fluorescence. A linear dependence of galactoside-binding rates on sugar concentration is observed in detergent, whereas reconstituted LacY binds substrate at a slower rate that is independent of sugar concentration. Rates of opening of the periplasmic cavity with LacY in detergent are independent of substrate concentration and are essentially the same for different galactosidic sugars. The findings demonstrate clearly that reconstituted LacY is oriented physiologically with a closed periplasmic side that limits access of sugar to the binding site. Moreover, opening of the periplasmic cavity is the limiting factor for sugar binding with reconstituted LacY and may be the limiting step in the overall transport reaction.
authors: Department of Physiology and Microbiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-7327, USA.
authors: Irina
authors: I
authors: Smirnova
authors: Vladimir
authors: V
authors: Kasho
authors: Junichi
authors: J
authors: Sugihara
authors: H Ronald
authors: HR
authors: Kaback
mesh_terms: D050260:Carbohydrate Metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009598:Nitrophenylgalactosides / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D050505:Mutant Proteins; D027981:Symporters; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C021499:lactose permease