Unmasking of an essential thiol during function of the membrane bound enzyme II of the phosphoenolpyruvate glucose phosphotransferase system of Escherichia coli.
The addition of N-ethylmaleimide (MalNEt), or of fluoro dinitrobenzene to a suspension of Escherichia coli during the phosphorylating uptake of methyl-alpha-D-glucopyranoside (Me-Glc), a glucose analog, stops uptake and phosphorylation and causes the loss of previously accumulated sugar and of its phosphate ester. After removal of the reagents, the phosphotransferase system remains irreversibly inactive. Pretreatment of the bacteria with the same reagents under the same conditions of concentration, pH, temperature and for the same length of time causes very little inactivation. Mercuric chloride, a reversible inactivator, prevents the phosphotransferase system from reacting simultaneously with MaINEt or with fluorodinitrobenzene. This protection strongly suggests that all three reagents react with the same site, presumably an -SH group. The change which makes this site available to the reagents depends on the phosphorylative uptake of Me-Glc. Preload of the cells and efflux of Me-Glc do not achieve the same change. The rate of inactivation is directly proportional to the rate of phosphorylative uptake. When the Km of phosphorylative uptake is modified by an uncoupling agent, the substrate concentration allowing half maximal rate of inactivation by MaINEt changes accordingly. The reactive sites of the phosphotransferase system can also be made accessible to the -SH group reagents by fluoride inhibition of phosphoenolpyruvate synthesis. This suggests that the inactivator resistent form is an "energized form" of the enzyme. The unmasking of the reactive site is not due to a change in transmembrane penetration of the reagents since incubation of toluene treated cells with MaINEt in the presence of phosphoenolpyruvate fails to inactivate the phosphotransferase activity, while incubation with MaINEt plus Me-Glc causes fast inactivation.
D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002701:Chloramphenicol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005947:Glucose; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D010728:Phosphoenolpyruvate; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013438:Sulfhydryl Compounds; D014050:Toluene / Q000494:pharmacology
D008757:Methylglucosides; D013438:Sulfhydryl Compounds; D014050:Toluene; D002701:Chloramphenicol; D010728:Phosphoenolpyruvate; D010770:Phosphotransferases; D005947:Glucose; D005033:Ethylmaleimide