The role of protons in the mechanism of galactoside transport via the lactose permease of Escherichia coli.
The kinetic mechanism of lactose transport across the cytoplasmic membrane has been investigated and the results related to standard models for the lactose-H+ symport reaction using computer simulation. It is shown that the biphasic kinetics reported for lactose uptake (Kaczorowski, G.J. and Kaback, H.R. (1979) Biochemistry 18, 3691-3697) are consistent with random binding of lactose and protons and rapid subsequent translocation of the ternary lactose-H+-permease complex. Such a model is also shown to explain the observed dependence of the kinetic parameters on the magnitude of the protonmotive force. Both sugar and protons are shown to cause product inhibition of lactose flux and the ability of standard models to account for the pattern of inhibition is discussed. Three apparent dissociation constants have been determined for the protonation reactions in the external medium: two (pKa 6.3 and 9.6) control the activity of the permease, whilst the third (pKa 8.3) controls the affinity of the permease for galactosides. A similar set of dissociation constants has been determined for the internal reactions. Again two (pKa 6 and 9.8) control activity and a third (pKa 8.8) controls the affinity for galactosides. The dissociation reactions characterised by pKa 8.3, 8.8, 9.6 and 9.8 are attributed to the dissociation of the substrate (symported) proton from the binary proton-permease complexes (pKa 8.3 and 8.8) and the ternary proton-galactoside-permease complexes (pKa 9.6 and 9.8). The third pair (pKa 6.3 and 6.0) must be interpreted as describing a separate protonation reaction which may have a regulatory or auxiliary role in transport.
D001426:Bacterial Proteins / Q000378:metabolism*; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005697:Galactosides; D006027:Glycosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; C021499:lactose permease; D007785:Lactose