Within the lactose permease, an arginine residue is found on a transmembrane segment at position 302. Based upon the effects of mutations at or in the vicinity of Arg-302, this residue has been implicated to be involved with H+ and/or sugar recognition. To further elucidate the role of this residue, we have substituted Arg-302 with serine, histidine, and leucine via site-directed mutagenesis. All three of these substitutions result in an impaired ability to transport galactosides as evidenced by their poor growth on minimal plates supplemented with lactose or melibiose. Furthermore, in vitro transport assays revealed substantial alterations in the kinetic constants for downhill lactose transport. The wild-type strain exhibited a K(m) for lactose transport of 0.30 mM and a V(max) of 267 nmol of lactose/min · mg of protein. The Ser-302, His-302, and Leu-302 were observed to have K(m) values of 0.18, 2.3, and 2.8 mM, and V(max) values of 11.6, 56.4, and 22.0 nmol of lactose/min · mg of protein, respectively. In uphill transport assays, all three mutants were unable to accumulate β-methyl-D- thiogalactoside. However, both the Ser-302 and His-302 mutants were able to accumulate lactose against a concentration gradient. During H+ transport assays, all three mutants were shown to transport H+ in conjunction with thiodigalactoside. In addition, the Ser-302 and His-302 strains exhibited small alkalinizations upon the addition of lactose. However, for the Leu-302 mutant, the addition of lactose did not result in a significant level of H+ transport. Finally, experiments were conducted which were aimed at measuring the ability of the mutant permeases to catalyze an H+ leak. In this regard, a comparison was made between the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses. The results of these experiments suggest that mutations at position 302 cause a sugar-dependent H+ leak.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1992|