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Reaction mechanism of phosphoglucosamine mutase from Escherichia coli
Abstract:
The phosphoglucosamine mutase (GlmM) from Escherichia coli, specifically required for the interconversion of glucosamine-6-phosphate and glucosamine-1-phosphate (an essential step in the pathway for cell-wall peptidoglycan and lipopolysaccharide biosyntheses) was purified to homogeneity and its kinetic properties were investigated. The enzyme was active in a phosphorylated form and catalysed its reaction according to a classical ping-pong bi-bi mechanism. The dephosphorylated and phosphorylated forms of GlmM could be separated by HPLC and coupled MS showed that only one phosphate was covalently 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 also capable of catalysing the interconversion of glucose-1-phosphate and glucose-6-phosphate isomers, although at a much lower (1400-fold) rate. Interestingly, the mutational change of the Ser100 to a threonine residue resulted in a 20-fold increase of the nonspecific phosphoglucomutase activity of GlmM, 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.
Insights
Phosphoglucosamine mutase (GlmM) from E. coli is essential for cell wall synthesis. This study identifies Ser102 as the phosphorylation site, crucial for GlmM activity and substrate specificity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Phosphoglucosamine mutase (GlmM) from Escherichia coli is vital for peptidoglycan and lipopolysaccharide biosynthesis.
- GlmM catalyzes the interconversion of glucosamine-6-phosphate and glucosamine-1-phosphate, a key step in these pathways.
Purpose of the Study:
- To purify and characterize the kinetic properties of E. coli GlmM.
- To identify the phosphorylation site and understand its role in enzyme activity and specificity.
Main Methods:
- Purification of GlmM to homogeneity.
- Kinetic analysis of enzyme activity.
- Separation of phosphorylated and dephosphorylated forms using HPLC.
- Mass spectrometry (MS) to identify the phosphorylation site.
- Site-directed mutagenesis (Ser100 to Threonine) to assess activity changes.
Main Results:
- GlmM was purified and found to be active in a phosphorylated form, operating via a ping-pong bi-bi mechanism.
- Mass spectrometry identified Ser102 as the single phosphorylation site on the enzyme.
- GlmM exhibited lower activity (1400-fold) for glucose-phosphate isomers.
- Mutation of Ser100 to Threonine increased non-specific phosphoglucomutase activity 20-fold.
Conclusions:
- The phosphorylation of Ser102 is critical for the catalytic activity of GlmM.
- The amino acid residue at position 100 (Serine or Threonine) influences the substrate specificity of hexosephosphate mutases.
- This finding provides insight into the molecular basis of enzyme specificity in sugar phosphate metabolism.