Facilitated diffusion of p-nitrophenyl-alpha-D-maltohexaoside through the outer membrane of Escherichia coli. Characterization of LamB as a specific and saturable channel for maltooligosaccharides.
LamB, an outer membrane protein of Escherichia coli, is a component of the maltose-maltooligosaccharide transport system. We used p-nitrophenyl-alpha-D-maltohexaoside, a chromogenic analog of maltohexaose, and a periplasmic amylase that hydrolyzes this compound to study the LamB-mediated diffusion of p-nitrophenyl-alpha-D-maltohexaoside into the periplasm. Using this approach, we were able to characterize LamB in vivo as a saturable channel for maltooligosaccharides. Permeation through LamB follows Michaelis-Menten kinetics, with a Km of 0.13 mM and a Vmax of 3.3 nmol/min/10(9) cells. Previous studies suggested that maltose-binding protein increases the rate of maltooligosaccharide diffusion through LamB. We show here that, at least in strains that are unable to transport maltooligosaccharides into the cytoplasm, maltose-binding protein does not influence the rate of substrate diffusion. The periplasmic amylase had been previously described as being of the alpha-type. We have now purified this protein and analyzed its mode of action using chromogenic maltooligosaccharides of varying length. Analysis of the hydrolytic products revealed that the enzyme recognizes its substrate from the nonreducing end and preferentially liberates maltohexaose, in contrast to the behavior of classical alpha-amylases that are endohydrolases. Using p-nitrophenyl-alpha-D-maltohexaoside as a substrate, we determined a Km of 3 microM and a Vmax of 0.14 mumol/min/mg of protein.
Freundlieb
S
S
Department of Biology, University of Konstanz, Federal Republic of Germany.
Ehmann
U
U
Boos
W
W
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism*; D010957:Plasmids
D001425:Bacterial Outer Membrane Proteins; D009844:Oligosaccharides; C021705:maltooligosaccharides