Role of protons in sugar binding to LacY

WT lactose permease of Escherichia coli (LacY) reconstituted into proteoliposomes loaded with a pH-sensitive fluorophore exhibits robust uphill H+ translocation coupled with downhill lactose transport. However, galactoside binding by mutants defective in lactose-induced H+ translocation is not accompanied by release of an H+ on the interior of the proteoliposomes. Because the pK(a) value for galactoside binding is similar to 10.5, protonation of LacY likely precedes sugar binding at physiological pH. Consistently, purified WT LacY, as well as the mutants, binds substrate at pH 7.5-8.5 in detergent, but no change in ambient pH is observed, demonstrating directly that LacY already is protonated when sugar binds. However, a kinetic isotope effect (KIE) on the rate of binding is observed, indicating that deuterium substitution for protium affects an H+ transfer reaction within LacY that is associated with sugar binding. At neutral pH or pD, both the rate of sugar dissociation (k(off)) and the forward rate (k(on)) are slower in D2O than in H2O (KIE is similar to 2), and, as a result, no change in affinity (K-d) is observed. Alkaline conditions enhance the effect of D2O on k(off), the KIE increases to 3.6-4.0, and affinity for sugar increases compared with H2O. In contrast, LacY mutants that exhibit pH-independent high-affinity binding up to pH 11.0 (e. g., Glu325 -> Gln) exhibit the same KIE (1.5-1.8) at neutral or alkaline pH (pD). Proton inventory studies exhibit a linear relationship between koff and D2O concentration at neutral and alkaline pH, indicating that internal transfer of a single H+ is involved in the KIE.