Effect of the Lipid Phase Transition on the Lactose Permease from Escherichia coli

The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 --> Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H(+) symport, Arrhenius plots with wild-type permease exhibit a discontinuity at 18-19 degrees C with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H(+) symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325 --> Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H(+) translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H(+) translocation rather than the conformational change(s) responsible for translocation across the membrane.