Coupling of alanine racemase and D-alanine dehydrogenase to active transport of amino acids in Escherichia coli B membrane vesicles.
Isolated membrane vesicles from Escherichia coli B grown on DL-alanine-glycerol carry out amino acid active transport coupled to D-alanine oxidation by a membrane-bound dehydrogenase. Several other D-amino acids are substrates for this D-alanine dehydrogenase and also drive concentrative uptake of solutes. Additionally, L-alanine and L-serine can energize solute transport by virtue of conversion to oxidizable D isomers by a membrane-bound alanine racemase. No other physiological L-amino acids were effective. Both membrane enzymes and consequent solute transport are markedly reduced in vesicles from glucose-grown cells. Respiratory chain uncouplers abolish the racemase-dehydrogenase-supported transport activity. When amino-oxyacetate at 10-4 M is added to the vesicles, the racemase activity and transport driven by L-alanine and L-serine is specifically and reversibly inhibited. D-Alanine-driven transport is unaffected. Similarly beta-chloro-L-alanine is an irreversible inactivator of the bound racemase but not the D-alanine dehydrogenase. Both the D and L isomers of beta-chloroalanine support oxygen uptake by the vesicles and initially stimulate L-(14C)proline active transport. However, oxidation of the beta-chloro-D-alanine rapidly uncouples active transport from substrate oxidation. This transport inactivation can be protected partially by dithiothreitol, putatively scavenging a reactive product of chloroalanine oxidation. Authentic beta-chloropyruvate produces the same transport uncoupling. When beta-chloro-L-alanine is employed as a substrate, no such transport inactivation is observed. This difference may stem from the possibility that the alanine racemase eliminates HCl from beta-chloro-L-alanine producing pyruvate, not the beta-chloropyruvate that would arise from racemization and then dehydrogenation. We have shown that exogenous pyruvate is oxidized by the vesicles and will also stimulate active transport of amino acids.
D000085:Acetates / Q000494:pharmacology; D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000591:Amino Acid Isomerases / Q000378:metabolism*; D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004140:Dinitrophenols / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D011392:Proline / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology; D013237:Stereoisomerism; D013997:Time Factors
D000085:Acetates; D000596:Amino Acids; D004140:Dinitrophenols; D006898:Hydroxylamines; D011773:Pyruvates; D011392:Proline; D000594:Amino Acid Oxidoreductases; D000591:Amino Acid Isomerases; D000409:Alanine; D004229:Dithiothreitol