The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H(+) transport (i.e., symport). While alternating access of sugar- and H(+)-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H(+) gradient ([Formula: see text]) functions kinetically by decreasing the K(m) for influx 50- to 100-fold with no change in K(d). The affinity of protonated LacY for sugar has an apparent pK (pK(app)) of ∼10.5, due specifically to the pK(a) of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H(+) efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pK(app) of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 ˗ Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the K(m) for efflux is almost insignificant relative to the 50- to 100-fold decrease in the K(m) for influx driven by ΔΨ (interior negative).