Delineating electrogenic reactions during lactose/H+ symport

Abstract

Electrogenic reactions accompanying downhill lactose/H+ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H+ translocation is abolished either by acidic pH or by a Glu325 → Ala mutation in the H+ binding site (this step with a rate constant of ∼200 s-1 for wild-type LacY leads to an intermediate proposed to represent an occluded state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ∼30 s-1 and a pK of 7.5. This partial reaction is assigned to rate-limiting H+ release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical-biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway. © 2010 American Chemical Society.