Reaction mechanism of phosphoglucosamine mutase from Escherichia coli

The phosphoglucosamine mutase (GlmM) from Escherichia coli, specifically re quired for the interconversion of glucosamine-6-phosphate and glucosamine-1 -phosphate (an essential step in the pathway for cell-wall peptidoglycan an d lipopolysaccharide biosyntheses) was purified to homogeneity and its kine tic properties were investigated. The enzyme was active in a phosphorylated form and catalysed its reaction according to a classical ping-pong bi-bi m echanism. The dephosphorylated and phosphorylated forms of GlmM could be se parated by HPLC and coupled MS showed that only one phosphate was covalentl y linked to the active site of the enzyme. The site of phosphorylation was clearly identified as Ser102 in the 445-amino acid polypeptide. GlmM was al so capable of catalysing the interconversion of glucose-l-phosphate and glu cose-6-phosphate isomers, although at a much lower (1400-fold) rate. Intere stingly, the mutational change of the Ser100 to a threonine residue resulte d in a 20-fold increase of the nonspecific phosphoglucomutase activity of G LmM, suggesting that the presence of either a serine or a threonine at this position in the consensus sequence of hexosephosphate mutases could be one of the factors that determines the specificity of these enzymes for either sugar-phosphate or amino sugar-phosphate substrates.