Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase.
In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.
Naught
Laura E
LE
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Regni
Catherine
C
Beamer
Lesa J
LJ
Tipton
Peter A
PA
D001665:Binding Sites; D002384:Catalysis; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008360:Mannosephosphates / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010733:Phosphoglucomutase / Q000235:genetics / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity; D013329:Structure-Activity Relationship
D005958:Glucosephosphates; D008360:Mannosephosphates; C027693:mannose-6-phosphate; D019298:Glucose-6-Phosphate; C031590:glucose-1-phosphate; D017875:Phosphotransferases (Phosphomutases); D010733:Phosphoglucomutase; C052799:phosphomannomutase