Kinetic mechanism and pH dependence of the kinetic parameters of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

Arch Biochem Biophys. 2001 Dec 1;396(1):111-8. doi: 10.1006/abbi.2001.2618.

Abstract

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is responsible for the formation of mannose 1-phosphate and glucose 1-phosphate in the human pathogenic bacterium Pseudomonas aeruginosa. Mannose 1-phosphate and glucose 1-phosphate are required for the biosynthesis of polysaccharides that contribute to the virulence of P. aeruginosa, so inhibitors of PMM/PGM may lead to clinically useful compounds. The V/K values for mannose 6-phosphate and glucose 6-phosphate show that they are equally good substrates for the enzyme. PMM/PGM overexpressed in Escherichia coli is isolated as a phosphoenzyme; surprisingly, mutation of serine 108 where phosphorylation occurs results in phosphorylation of a different residue so that activity is reduced only 20-fold from that of wild-type enzyme. In the reverse reaction glucose 1-phosphate exhibits substrate inhibition, which arises through its competition with the activator glucose 1,6-bisphosphate for binding to dephosphoenzyme. This phenomenon is consistent with a mechanism in which the enzyme phosphorylates the substrate to generate a bisphosphorylated intermediate that reorients in the active site to return its original phosphoryl group to the enzyme and generate the observed product. The pH dependence of the kinetic parameters suggests that the active site contains a residue that serves as a general base in the catalytic reaction and one that acts as a general acid. However, the pK of the general acid is 7.4 and that of the general base is 8.4 so these residues exist in a state of reverse protonation in the active enzyme.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alginates / metabolism
  • Glucose-6-Phosphate / metabolism
  • Glucosephosphates / metabolism
  • Glucuronic Acid
  • Hexuronic Acids
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mannosephosphates / metabolism
  • Models, Chemical
  • Phosphoglucomutase / metabolism*
  • Phosphotransferases (Phosphomutases) / metabolism*
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / pathogenicity

Substances

  • Alginates
  • Glucosephosphates
  • Hexuronic Acids
  • Mannosephosphates
  • mannose-6-phosphate
  • Glucose-6-Phosphate
  • Glucuronic Acid
  • glucose-1-phosphate
  • Phosphotransferases (Phosphomutases)
  • Phosphoglucomutase
  • phosphomannomutase