The Energy‐Coupling Controlled Efflux of 2‐Keto‐3‐deoxy‐d‐gluconate in Escherichia coli K 12

Experiments were devised to test the plausibility and the predictions of an efflux rate equation which was previously derived [10]. 1 2-Keto-3-deoxy-d-gluconate transport system conforms with universal laws relating zero-trans influx, influx at steady-state, steady-state levels of accumulation to external and internal substrate concentrations. 2 Full-time-course uptake kinetics fit the linearized graphical representation that can be inferred from the integrated rate equation. 3 Influx does not depend upon internal substrate concentration nor upon energy-coupling. 4 Zero-trans outflux (leak into empty medium) is a first-order process (rate constant:0.02 min−1) and not mediated by the carrier. Absence of cis-competition with d-glucuronate is in agreement with a simple diffusion mechanism. 5 Outflux increases when external substrate concentration is raised (counterflow). Outflux at steady-state equilibrates influx, and is a first-order process (rate constant: 0.15 min−1). 6 Uncoupling with azide leads to accelerate zero-trans outflux by a factor of 2–3. No further acceleration is obtained when other classical uncouplers are used. The process remains first-order, independent of the amount of carrier, and is accelerated by the presence of internal d-glucuronate as a result from trans-inhibition of the recapture. 7 Exchange outflux is all the more accelerated by azide as the carrier is less saturated. The process is clearly carrier-mediated and the outflux rate obeys a Michaelis law with respect to internal concentration. V is equal to V for influx. 8 Homo and hetero-overshoot experiments are in agreement with the participation of the carrier for mediating influx as well as outflux. 9 The kinetics of d-glucuronate outflux in a strain lacking the specific hexuronate permease but carrying the 2-keto-3-deoxy-d-gluconate permease are similar to those obtained with 2-keto-3-deoxy-d-gluconate. We draw the conclusion that energy-coupling promotes the adjustment of outflux without interfering with influx rate. It apparently acts by reducing, in a continuous range, the affinity of the carrier facing inwards. The discussion is focused on the comparison with previously published models and on possible molecular mechanisms.