A Transport System for 2‐Keto‐3‐deoxy‐d‐gluconate Uptake in Escherichia coli K12

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Abstract
We have presented kinetic evidence for the occurrence in Escherichia coli K 12 of an active transport system responsible for 2‐keto‐3‐deoxy‐d‐gluconate uptake. It has been studied in constitutive strains PK4 and CAK 101 since it is completely cryptic in the wild‐type strain. The accumulation is temperature‐dependent, coupled to electron and energy‐yielding reactions, and is inhibited by thiol reagents. 2‐Keto‐3‐deoxy‐d‐gluconate is not transported through the phospho‐enolpyruvate‐transferase system. Entry reaction displays saturation kinetics obeying the Michaelis‐Menten laws. The Km value has been found to be 0.25 ± 0.05 mM (at 30 °C and pH 7.0), by measuring initial rates of entrance as well as steady‐state levels. Both the operator‐constitutive PK4 strain and the regulator‐constitutive CAK101 strain exhibit the same affinity but apparent V are 10 and 20 nmol × mg‐1× min‐1, respectively. The 2‐keto‐3‐deoxy‐d‐gluconate permeation system is endogenously induced in a strain lacking 2‐keto‐3‐deoxy‐d‐gluconate kinase, when grown on any intermediary product of the hexuronide catabolic pathway, able to enter the cell and anterior to 2‐keto‐3‐deoxy‐d‐gluconate in the sequence. We presented further evidence making it very likely that 2‐keto‐3‐deoxy‐d‐gluconate itself is the true inducer. The constitutive synthesis of the transport system is sensitive to the catabolite repression exerted by glucose and pyruvate. By testing a wide number of sugars and derivatives as possible substrates, only d‐glucuronate is a strictly competitive inhibitor (Ki= 1.5 mM). Since a specific hexuronate permease has been described elsewhere, we were led to demonstrate that d‐glucuronate could be taken up through both its own specific system with high affinity and through the 2‐keto‐3‐deoxy‐d‐gluconate specific system with lower affinity. The exit reaction rate is first oder with respect to internal concentration, temperature‐dependent (exit rate constant kex is 0.0180 min‐1 at 30 °C) but is not carrier‐mediated.