Mechanism of Lactose Transport in Escherichia coli Membrane Vesicles: Evidence for the Involvement of Histidine Residue(s) in the Response of the lac Carrier to the Proton Electrochemical Gradient

Exposure of Escherichia coli ML 308-225 membrane vesicles to diethyl pyrocarbonate or to light in the presence of rose bengal and air causes inactivation of active lactose transport and/or counterflow in a manner that is blocked by substrates of the lac carrier protein. The effect of pH on inactivation indicates that loss of activity is due to modification of an amino acid residue with a pK_a between 6.0 and 6.5, and the pH profiles for inactivation are very similar to those observed for the reaction of histidine. Furthermore, titration experiments with diethyl pyrocarbonate imply that acylation of a single site in the β-galactoside transport system is sufficient for inactivation. In contrast, neither reagent inhibits the ability of the carrier to bind p-nitrophenyl α-D-galactopyranoside nor its ability to catalyze facilitated diffusion of lactose [Padan, E., Patel, L., & Kaback, H. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6221]. Experiments carried out over an extended range of lactose concentrations demonstrate that treatment with diethyl pyrocarbonate causes the lac transport system to exhibit biphasic kinetics. One component of the overall process exhibits kinetic parameters typical of active transport (i.e., low apparent K_m), and the other has the characteristics of facilitated diffusion (i.e., high apparent K_m). Since partial dissipation of the proton electrochemical gradient leads to similar biphasic kinetics [Robertson, D. E., Kaczorowski, G. J., Garcia, M.-L., & Kaback, H. R. (1980) Biochemistry 19, 5692] and treatment of the vesicles with diethyl pyrocarbonate induces a similar alteration with no reduction in the proton electrochemical gradient, it is suggested that a histidine residue(s) in the lac carrier is (are) involved in the response of the protein to the proton gradient. In the following paper [Patel, L., Garcia, M. L., & Kaback, H. R. (1982) Biochemistry (following paper in this issue)], the effects of diethyl pyrocarbonate on lactose-induced proton movements in isolated membrane vesicles are examined.