Melibiose permease of Escherichia coli. Characteristics of co-substrates release during facilitated diffusion reactions

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was investigated by further analyzing the Na+ (H+ or Li+)-coupled facilitated diffusion reactions catalyzed by the carrier in de-energized membrane vesicles, with particular emphasis on the reaction of sugar exchange at equilibrium. It is first shown that melibiose exchange at equilibrium proceeds without concomitant movement of Na+, i.e. the coupled cation is kinetically occluded during the melibiose exchange reaction. These results provide further experimental support for the model of Na+ sugar co-transport of the physiological substrate melibiose previously suggested (Bassilana, M., Pourcher, T., and Leblanc, G. (1987) J. Biol. Chem. 262, 16865-16870) in which: 1) the mechanisms of co-substrate binding to (or release from) the carrier are ordered processes on both the outer (Na+ first, sugar last) and inner membrane surfaces (sugar first, Na+ last) and give rise to a mirror-type model; 2) release of Na+ from the carrier on the inner membrane surface is very slow and rate-limiting for carrier cycling but is fast on the opposite side, contributing to the asymmetrical functioning of the permease. On the other hand, analysis of the exchange of identical sugars (homologous exchange) and different sugar analogs (heterologous exchange) indicates that the overall rate of sugar exchange reaction coupled to Na+ or Li+ is limited by the rate of one (or more) partial step(s) associated with the inflow of co-substrates and most probably by the rate of sugar release into the intravesicular medium. It is proposed that the variability of the facilitated diffusion reactions catalyzed by the carrier in the presence of different coupled cations and/or sugar analogs reflects variations in the rate of co-substrate release from the carrier on the inner membrane surface.