The effects of N-ethylmaleimide on active amino acid transport in Escherichia coli

N-Ethylmaleimide (MalNEt) binds covalently and without specificity to accessible sulfhydryl residues in proteins. In some cases specificity has been imposed on this reaction by manipulating reaction conditions, yielding information concerning both enzyme mechanism and the identity of specific proteins (for example C.F. Fox and E.P. Kennedy (1965) Proc. Natl. Acad. Sci. U.S. 54, 891–899) and R.E. McCarty and J. Fagan (1973) Biochemistry 12, 1503–1507). We have examined the effects of MalNEt on the active accumulation of nine amino acids by Escherichia coli strains ML 308-225 and DL 54. Whole cells have been used in order that transport systems both dependent on and independent of periplasmic binding proteins could be studied under various conditions of energy supply for transport. Our results suggest that the systems transporting ornithine, phenylalanine and proline are those most likely to undergo inactivation by direct reaction of MalNEt with the transport apparatus, rather than merely via side effects such as interruption of their energy supply. The inhibition of proline transport is specifically enhanced by the presence of proline, competitive inhibitors of proline transport, or carbonylcyanide p-trifluoromethoxyphenylhydrazone during MalNEt treatment. The other eight systems tested showed no analogous effects.     

The reaction of carbonyl cyanide phenylhydrazones with thiols

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and amino-thiols (cysteine, glutathione) to give corresponding N-(substituted phynyl)-N′-(alkythiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular Ad_N type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS^? ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group).The reactivity of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone < carbonyl cyanide m-chlorophenylhydrazone < carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pK_a constants. On the other hand, the reactivity of thiols increases with their basicity.The reactivity of carbonyl cyanide phynylhydrazone with thiols is comparable with the reactivity of phynyl isothiocyanate and N-ethylmaleimide. It was demostrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12).The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.     

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-α-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 MalNEt 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 K_m of phosphorylative uptake is modified by an uncoupling agent, the substrate concentration allowing half maximal rate of inactivation by MalNEt 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 MalNEt in the presence of phosphoenolpyruvate fails to inactivate the phosphotransferase activity, while incubation with MalNEt plus Me-Glc causes fast inactivation.