title: Kinetic properties of purified Pseudomonas aeruginosa phosphorylcholine phosphatase indicated that this enzyme may be utilized by the bacteria to colonize in different environments.
abstract: Pseudomonas aeruginosa phosphorylcholine phosphatase from periplasmic extracts of bacteria grown on choline as the sole carbon and nitrogen source was purified to homogeneity. The enzyme represented nearly 1% of the total protein found in the periplasmic space and is a monomer of approximately 53 kDa with an isoelectric point of 7.5. The optimum pH with phosphorylcholine was in the range of 5-8; with phosphorylethanolamine there was a peak at pH 6, and with p-nitrophenyl-phosphate (p-NPP) the optimum was at pH 5. Studies carried out at pH 5 indicated: i) For the three substrates above, Mg2+, Zn2+, or Cu2+ was necessary for maximal activity. ii) With p-NPP, these cations bound to the free enzyme in an ordered bireactant system. iii) With phosphorylethanolamine, Mg2+, Zn2+, or Cu2+ bound to the free enzyme in an at random bireactant system. iv) With phosphorylcholine, maximal activity was obtained with cation concentrations as low as 100 nM. v) Al3+ ions were inhibitors of the enzyme activity. The n (Hill coefficient) values for the inhibition by Al3+ with phosphorylcholine or p-NPP were 1 or 4, respectively. vi) The enzyme exhibited two affinity sites for phosphorylcholine. With Mg2+, a site with a Km value of 0.5 mM was detected; the corresponding Vmax was 25 micromol min-1 (mg protein)-1; without Mg2+, the enzyme displayed Km and Vmax values of 0.09 mM and 4.2 micromol min-1 (mg protein)-1, respectively. Studies carried out at pH 7.4 indicated: i) The enzyme could not catalyze the hydrolysis of p-NPP, and phosphorylethanolamine was a poor substrate in either the presence or absence of divalent cations. ii) The enzyme activity measured with phosphorylcholine was independent of divalent cations or was not inhibited by Al3+ ions. iii) With or without Mg2+, the enzyme exhibited two affinity sites for phosphorylcholine; the Km values were 0.05 mM and 0.5 mM with their corresponding Vmax of 5.6 and 25 micromol min-1 (mg protein)-1, respectively.
authors.affiliation: Química Biológica, Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, 5800 Río Cuarto, Córdoba, Argentina.
authors.forename: M A
authors.initials: MA
authors.lastname: Salvano
authors.forename: C E
authors.initials: CE
authors.lastname: Domenech
mesh_terms: D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010767:Phosphorylcholine / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
chemical_list: D010767:Phosphorylcholine; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D002794:Choline