Effect of the lipid phase transition on the lactose permease from Escherichia coli

The temperature dependence of lactose active transport, efflux down a conce ntration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose perme ase and mutant Glu325 --> Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve Hf symport, Arrhenius plots with wild-type permease exhibit a discontinuity at 18-19 degreesC wit h a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve Ht symport, the change in acti vation energy is much less pronounced (2-3-fold) than that observed for act ive 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 Hf translocation, exhibits no cha nge whatsoever in activation energy. The findings are consistent with the c onclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and Hf translocation rather than the conformati onal change(s) responsible for translocation across the membrane.