title: Reversible inactivation of vectorial phosphorylation by hydroxybutynoate in Escherichia coli membrane vesicles.
abstract: The acetylenic hydroxy acid 2-hydroxy-3-butynoate causes irreversible inactivation of the Escherichia coli membrane-bound flavoenzyme D-lactic dehyrogenase, and thus blocks D-lactate dependent active transport in isolated membrane vesicles [Walsh, C. T., Abeles, R. H., and Kaback H. R. (1972), J. Biol. Chem. 247, 7858]. The inactivator is a suicide substrate for the dehydrogenase, undergoing a small number of turnovers before partitioning between oxidation and inactiviation. It is now demonstrated that reactive product molecules of 2-keto-3-butynoate can diffuse in the membranes to a component of the phosphotransferase system and cause time-dependent and covalent inactivation of phosphoenolpyruvate-dependent hexose uptake. Membrane vesicles from double mutants with low levels of both D- and L-lactic dehydrogenase lose only 30 percent of their hexose uptake capacity on exposure to hydroxybutynoate under conditions sufficient to fully inactivate hexose transport in wild type vesicles. Transport of 1-[14C]hydroxybutynoate into vesicles is followed by rapid covalent labeling of membrane proteins by the reactive, enzymatically generating keto acid oxidation product. Incubation of hydroxybutynoate-inactivated vesicles (5% residual activity) for 20 min in buffer with 10 mM dithiothreitol results in reactivation of 63% of the hexose transport activity, a 12-fold increase in activity. No reactivation occurs if the vesicular phosphotransferase system is inactivated by keto acid derived from membrane oxidation of the olefinic congener 2-hydroxy-3-butenoate. In contrast to thiol reactivation of acetylenic-blocked glucose transport, blockage of D-lactate-stimulated proline uptake is not alleviated, stressing different modes of inactivation of the phosphotransferase system compared to the membranous lactate dehydrogenases.
mesh_terms: D001693:Biological Transport, Active; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006601:Hexoses / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000494:pharmacology; D009154:Mutation; D010770:Phosphotransferases / Q000378:metabolism*
chemical_list: D006601:Hexoses; D006885:Hydroxybutyrates; D007773:Lactates; C002479:2-hydroxy-3-butynoic acid; D007770:L-Lactate Dehydrogenase; D010770:Phosphotransferases; D004229:Dithiothreitol