Different Effects of Mg2+ and Zn2+ on the Two Sites for Alkylammonium Compounds in Pseudomonas aeruginosa Phosphorylcholine Phosphatase

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho), is activated by Mg2+ or Zn2+, and is inhibited by high concentrations of substrate. This study has shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and the other in an inhibitory site responsible for the binding of the alkylammonium moiety. The catalytic mechanism for the entry of Pcho in both sites and Zn2+ or Mg2+ follows a random sequential mechanism. However, Zn2+ is more effective than Mg2+ at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. In contrast, Mg2+ produces a relaxed or open conformation. 1. Introduction Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho) [1]. Pcho is the product of the action of hemolytic phospholipase C (PlcH) on phosphatidylcholine or sphingomyelin and is hydrolyzed to choline and inorganic phosphate (Pi) by the action of PchP. Thus, both the PlcH and PchP enzymes are involved in the pathogenesis of P. aeruginosa [2]. PchP contains three motifs that are characteristic of the enzymes belonging to the haloacid dehalogenase (HAD) superfamily [3]. Moreover, all three motifs have an important role in the catalytic process of Pcho or p-nitrophenylphosphate (p-NPP) in the presence of Mg2+, Zn2+, or Cu2+ as activators of the enzyme [4]. Using Pcho as the substrate, we have shown that Mg2+ is an equal activator for the enzyme at pH 5.0 and at pH 7.4; however, Zn2+ is an activator at pH 5.0 but an inhibitor at pH 7.4. The inhibition produced by Zn2+ at pH 7.4 is reversible and occurs in the presence or absence of Mg2+. This activation or inhibition of PchP by Zn2+ is caused by the transition from octahedral to tetrahedral geometry in the coordination sphere of the metal ion [5]. These results, in combination with the fact that PchP is inhibited by high Pcho concentrations and previous observations that different AACs may act as inhibitors of PchP [1, 6, 7], led us to evaluate the catalytic mechanism of PchP with Pcho as the substrate, Mg2+ or Zn2+ as activators, and AACs as inhibitors. 2. Materials and Methods 2.1. Materials Isopropyl-β-D-thiogalactopyranoside (IPTG) and HisLinkTM resin were purchased from Promega. Pcho and p-NPP were purchased from