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

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is responsible f or the formation of mannose 1-phosphate and glucose 1-phosphate in the huma n pathogenic bacterium Pseudomonas aeruginosa Mannose 1-phosphate and gluco se I-phosphate are required for the biosynthesis of polysaccharides that co ntribute to the virulence of P. aeruiginosa, so inhibitors of PMM/PGM may l ead 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 phosphoe nzyme; surprisingly, mutation of serine 108 where phosphorylation occurs re sults in phosphorylation of a different residue so that activity is reduced only 20-fold from that of wild-type enzyme. In the reverse reaction glucos e 1-phosphate exhibits substrate inhibition, which arises through its compe tition with the activator glucose 1,6-bisphosphate for binding to dephospho enzyme. This phenomenon is consistent with a mechanism in which the enzyme phosphorylates the substrate to generate a bisphosphorylated intermediate t hat reorients in the active site to return its original phosphoryl group to the enzyme and generate the observed product. The pH dependence of the kin etic parameters suggests that the active site contains a residue that serve s as a general base in the catalytic reaction and one that acts as a genera l 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 th e active enzyme. (C) 2001 Elsevier Science.