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.
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.
D000332:Aerobiosis; D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism*; D000693:Anaerobiosis; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology; D013237:Stereoisomerism
D000596:Amino Acids; D008757:Methylglucosides; D011773:Pyruvates; D000409:Alanine