Stereochemical course of reactions catalyzed by the bacterial phosphoenolpyruvate: glucose phosphotransferase system

Abstract

The overall stereochemical course of the reactions leading to the phosphorylation of methyl .alpha.-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system was investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl .alpha.-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). Sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group was transferred at least 5 times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in 2 steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, these results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.