Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding.
The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction. Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants. Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding. In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3. This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside. The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover. In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced. The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water. The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside. In a strain which carries the YUN mutation (Wilson, T.H. and Kusch, M. (1972) Biochim. Biophys. Acta 255, 786-797), two reactive thiols were characterised. The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water. The less reactive thiol appears to be more deeply buried than that of the native protein. Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.
D001693:Biological Transport, Active / Q000187:drug effects; D003545:Cysteine / Q000502:physiology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011487:Protein Conformation; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters
D029968:Escherichia coli Proteins; D005697:Galactosides; D006027:Glycosides; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C021499:lactose permease; D003545:Cysteine