Kinetic Properties of Purified Pseudomonas aeruginosa Phosphorylcholine Phosphatase Indicated That This Enzyme May Be Utilized by the Bacteria to Colonize in Different Environments (original) (raw)

1999, Current Microbiology

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, Mg 2ϩ , Zn 2ϩ , or Cu 2ϩ was necessary for maximal activity. ii) With p-NPP, these cations bound to the free enzyme in an ordered bireactant system. iii) With phosphorylethanolamine, Mg 2ϩ , Zn 2ϩ , or Cu 2ϩ 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) Al 3ϩ ions were inhibitors of the enzyme activity. The n (Hill coefficient) values for the inhibition by Al 3ϩ with phosphorylcholine or p-NPP were 1 or 4, respectively. vi) The enzyme exhibited two affinity sites for phosphorylcholine. With Mg 2ϩ , a site with a K m value of 0.5 mM was detected; the corresponding V max was 25 µmol min Ϫ1 (mg protein) Ϫ1 ; without Mg 2ϩ , the enzyme displayed K m and V max values of 0.09 mM and 4.2 µmol min Ϫ1 (mg protein) Ϫ1 , 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 Al 3ϩ ions. iii) With or without Mg 2ϩ , the enzyme exhibited two affinity sites for phosphorylcholine; the K m values were 0.05 mM and 0.5 mM with their corresponding V max of 5.6 and 25 µmol min Ϫ1 (mg protein) Ϫ1 , respectively.