Abstract
Abstract The alkenoic hydroxyacid, vinylglycolate (2-hydroxy-3-butenoic acid), inactivates Enzyme I of the P-enolpyruvate-P-transferase system in Escherichia coli. By this means, vinylglycolate is a potent, irreversible inactivator of vectorial phosphorylation in whole cells and membrane vesicles prepared from this organism. Evidence is presented indicating that prior to inactivation of the P-transferase system, the unsaturated hydroxy acid gains access to the intravesicular pool by means of a lactate transport system. Subsequently, the compound is oxidized by membrane-bound, flavin-linked d- and l-lactate dehydrogenases to yield a reactive electrophile (presumably 2-keto-3-butenoate) which reacts with Enzyme I and possibly other cellular components. The following observations support this interpretation: 1. Vectorial phosphorylation catalyzed by whole cells and isolated membrane vesicles undergoes a time-dependent, irreversible inactivation in the presence of vinylglycolate. Vinylacetate (3-butenoate) has no effect. 2. Membrane vesicles prepared from a d-lactate dehydrogenase mutant undergo inactivation of vectorial phosphorylation at a slower rate. 3. Reagents or conditions which block d- and l-lactate dehydrogenase activity or electron transfer diminish the rate of inactivation of the P-transferase system in isolated membrane vesicles. Similarly, the rate of inactivation is diminished by addition of d- or l-lactate, or both, to the reaction mixtures. 4. Nucleophilic reagents such as dithiothreitol and histidine methylester protect against inactivation of P-enolpyruvate-dependent sugar uptake in isolated membrane vesicles. 5. Vinylglycolate is a competitive, reversible inhibitor of respiration-linked d-lactate transport in membrane vesicles, but does not inactivate the transport of lactose, proline, or succinate. 6. Treatment of whole cells with vinylglycolate results in inactivation of Enzyme I activity. Enzyme II and HPr activity remain intact, and vesicles treated with vinylglycolate exhibit normal Enzyme II activity. Additional experiments demonstrate that vinylglycolate is a potent inhibitor of growth in E. coli. At 1 µm vinylglycolate, the rate of growth is inhibited by approximately 50%; at 10 µm by over 95%; and at 100 µm, growth is inhibited completely.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have