JBC, Vol. 250, Issue 23, 8921-8930, Dec, 1975

Active transport in Escherichia coli B membrane vesicles. Differential inactivating effects from the enzymatic oxidation of beta-chloro-L-alanine and beta-chloro-D-alanine

G. Kaczorowski, L. Shaw, R. Laura and C. Walsh

Isolated membrane vesicles from Escherichia coli B grown on DL-alanine and glycerol carry out amino acid active transport coupled to a membrane-bound D-alanine dehydrogenase (Kaczorowski, G., Shaw, L., Fuentes, M., and Walsh, C. (1975) J. Biol. Chem. 250, 2855). Certain L-amino acids can also energize solute transport by conversion to their D isomers via an alanine reacemase. Both D-chloroalanine and L-chloroalanine initially drive amino acid and methyl-beta-thiogalactose uptake. The D isomer however causes rapid inactivation of both dehydrogenase-coupled transport and the phosphotransferase system. Transport functions can be protected by dithiothreitol which is postulated to act as a scavenging nucleophile. This inactivation by the D isomer is time-dependent and irreversible not only for proline transport but also for alpha-methylglucoside uptake. Unlike the D isomer, beta-chloro-L-alanine does not inactivate transport. L-Chloroalanine is not racemized to the D isomer but rather undergoes a racemase catalyzed beta elimination of chloride ion to produce pyruvate. Pyruvate can subsequently be oxidized to stimulate active transport. This pyridoxal phosphate-dependent racemase is inactivated by low concentrations of D-chloroalanine but the L isomer can only cause inactivation at a 40-fold higher concentration and longer times of exposure. The D-alanine dehydrogenase-catalyzed oxidation product of D-chloroalanine is chloropyruvate, and this keto acid is hypothesized to be the inactivating species of transport for the following reasons. Chloropyruvate has been isolated from D-chloroalanine oxidation but not from oxidation of the L isomer. Chlorolactate which can be oxidized to chloropyruvate (via membrane-bound lactate dehydrogenases) also causes inactivation of transport in E. coli K-12 membrane vesicles. Mutants having diminished lactate dehydrogenase activity show a slower rate of inactivation with chlorolactate. Moreover, synthetic chloropyruvate irreversibly inactivates both active transport of proline and phosphotransferase system-dependent group translocation of alpha-methylglucoside. The effects of D- and L-chloroalanine and chlorolactate on transport in membrane vesicles are also seen in whole cells.
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