KINETIC-ANALYSIS OF LACTOSE AND PROTON COUPLING IN GLU(379) MUTANTS OF THE LACTOSE TRANSPORT PROTEIN OF STREPTOCOCCUS-THERMOPHILUS

The role of Glu(379) in the lactose-H+ symport protein (LacS) of Strep tococcus thermophilus was studied by analyzing the kinetic mechanism o f transport of wild-type and Ala(379), Asp(379), and Gln(379) mutant p roteins. Glu(379) forms part of the sequence motif Lys-X-X-His-X-X-Glu that is present in a number of sugar transport proteins, including La cY of Escherichia coli. The E379A and E379Q mutants were defective in the uptake of lactose against a concentration gradient and lactose dep endent 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 E879D mutant was partially defect ive in the coupled transport of lactose and protons. These results sug gest that an acidic residue at position 379 is required for the couple d uptake of lactose and protons and are consistent with a mechanism in which lactose transport in the E379A and E379Q mutants occurs by unip ort rather than proton symport. Lactose efflux down a concentration gr adient in wild-type LacS and LacS-E379D increased with pH with apparen t pK (pK(alpha)) values of greater than or equal to 8.5 and 8.0, respe ctively, whereas efflux in the E379Q mutant increased sigmoidally with a pK(alpha) of about 6.0. Imposition of an artificial membrane potent ial (inside negative) in membrane vesicles bearing wild-type LacS or L acS-E379Q not only inhibited the lactose efflux mediated by wild-type but also that of the mutant enzyme. To associate the role of Glu(379) with specific step(s) in the translocation cycle of LacS, the properti es of wild-type LacS and the Glu(379) mutants have been evaluated by n umerical analysis of simple kinetic schemes for translocation catalysi s by solute H+ symport proteins. The properties of the wild-type enzym e are consistent with a mechanism in which the order of ligand binding on the inside is substrate first and proton last, whereas the order i s random (or proton first, substrate last) at the outer surface of the membrane. The wild-type enzyme is asymmetric with regard to proton bi nding; the pK for proton binding on the outside is at least 4 units hi gher than the pK on the inside. The properties of the Glu(379) mutants correspond with a lowering of the pK on the outside (pK(OUT) similar to pK(IN)), and the induction of a leak pathway in which the binary en zyme-substrate complex becomes mobile.