Dependence on pH of parameters of lactose transport in Escherichia coli. Evidence for an essential protonated group of the carrier.

The kinetic parameters Km and V of transported by the lactose permease of Escherichia coli have been explored in the pH range 4.8--9.2. Besides uphill transport of methylthiogalactoside, two other criteria have been used. Downhill transport of o-nitrophenylgalactoside and substrate protection of the carrier against thiol reagents have both been explored in normal aerated cells and in cells inhibited by cyanide plus azide, therefore unable to build up a proton-motive force. V of the transport processes did not exhibit a major pH dependence that would support an essential protonation. Ktransport for methylthiogalactoside and for o-nitrophenylgalactoside in the energized and in the inhibited state did not show a sharp pH dependence between pH 4.8 and 8.0, but increased between pH 8 and 9, as would be expected if there were an essential protonated group with a pK of 8--8.4, depending on the test utilized. Substrate protection allowed the calculation of a Kprotection which was close to the corresponding Ktransport and was also largely independent of pH between 5 and 8 and independent of energy supply. The role of energization in substrate-carrier binding and the role of the essential protonation in the context of the proton symptom model are discussed.