The glucose‐specific carrier of the Escherichia coli phosphotransferase system

Abstract

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EII Glc ) of the Escherichia coli phospho enol pyruvate–sugar phosphotransferase system (PTS). 2′,3′‐Epoxypropyl β‐ d ‐glucopyranoside ( 3a ) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EII Glc but not pseudosubstrates. They are selective for EII Glc as demonstrated by comparison with EII Man , another Glc‐specific but structurally different transporter. 3a is the only analogue that inhibits EII Glc by binding to the high‐affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro , methyl 6,7‐anhydro‐ d , l ‐glycero‐α‐ d ‐gluco‐heptopyranoside ( 1d ), preferentially interacts with the low‐affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EII Glc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EII Glc is rapidly inactivated by the 6‐ O ‐bromoacetyl esters of methyl α‐ d ‐glucopyranoside ( 1a ) and methyl α‐ d ‐mannopyranoside ( 1c ), methyl 6‐deoxy‐6‐isothiocyanato‐α‐ d ‐glucopyranoside ( 1e ), β‐ d ‐glucopyranosyl isothiocyanate ( 3c ) and β‐ d ‐glucopyranosyl phenyl isothiocyanate ( 3d ). Phosphorylation of EII Glc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EII Glc for inactivation by 1e and 3d , which is interpreted as the effect of glucose‐induced conformational changes in the dimeric transporter. Glc also sensitizes EII Glc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EII Glc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.