N-ethylmaleimide (NEM) inhibits lactose uptake in E. coli by reacting with the M protein component of the lac permease system. In an attempt to estimate the distance between the NEM reactive site and the substrate binding site, we have synthesized a βgalactoside with NEM as the aglycon moiety (NEM-gal). NEM-gal was a more effective inhibitor of lactose transport than was NEM. Part of the inhibition by NEM-gal was caused by competition with lactose for the substrate binding site. To estimate this part of the inhibition, we synthesized the saturated and thus the unreactive N-ethylsuccinimide (NES) analog of NEM-gal. NES-gal was a competitive inhibitor of lactose uptake. The remainder of the inhibition by NEM-gal followed first-order kinetics with the same rate constant as NEM. In addition, the protective effect of thiodigalactoside against the inhibition of transport by NEM was also observed against irreversible inhibition by NEM-gal. We suggest that the reactivity of NEM was unaltered by bringing it near the βgalactoside binding site by way of covalent attachment to galactose. We conclude that the distance between the NEM reactive site and the position of the glycosidic oxygen of βgalactosides bound to the lactose site is greater than 8Å.
- (N-ethylmaleimide sugar analogue)
- Lactose transport
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology