Kinetic analysis of lactose and proton coupling in Glu379 mutants of the lactose transport protein of Streptococcus thermophilus.

The role of Glu379 in the lactose-H+ symport protein (LacS) of Streptococcus thermophilus was studied by analyzing the kinetic mechanism of transport of wild-type and Ala379, Asp379, and Gln379 mutant proteins. Glu379 forms part of the sequence motif Lys-X-X-His-X-X-Glu that is present in a number of sugar transport proteins, including LacY of Escherichia coli. The E379A and E379Q mutants were defective in the uptake of lactose against a concentration gradient and lactose-dependent proton uptake, but catalyzed facilitated influx of lactose down a concentration gradient and equilibrium exchange with rates similar to that of the wild-type enzyme. The E379D mutant was partially defective in the coupled transport of lactose and protons. These results suggest that an acidic residue at position 379 is required for the coupled uptake of lactose and protons and are consistent with a mechanism in which lactose transport in the E379A and E379Q mutants occurs by uniport rather than proton symport. Lactose efflux down a concentration gradient in wild-type LacS and LacS-E379D increased with pH with apparent pK (pKa) values of > or = 8.5 and 8.0, respectively, whereas efflux in the E379Q mutant increased sigmoidally with a pKa of about 6.0. Imposition of an artificial membrane potential (inside negative) in membrane vesicles bearing wild-type LacS or LacS-E379Q not only inhibited the lactose efflux mediated by wild-type but also that of the mutant enzyme. To associate the role of Glu379 with specific step(s) in the translocation cycle of LacS, the properties of wild-type LacS and the Glu379 mutants have been evaluated by numerical analysis of simple kinetic schemes for translocation catalysis by solute H+ symport proteins. The properties of the wild-type enzyme are consistent with a mechanism in which the order of ligand binding on the inside is substrate first and proton last, whereas the order is random (or proton first, substrate last) at the outer surface of the membrane. The wild-type enzyme is asymmetric with regard to proton binding; the pK for proton binding on the outside is at least 4 units higher than the pK on the inside. The properties of the Glu379 mutants correspond with a lowering of the pK on the outside (pKOUT approximately pKIN), and the induction of a leak pathway in which the binary enzyme-substrate complex becomes mobile.