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Different Effects of Mg and Zn on the Two Sites for Alkylammonium Compounds in Pseudomonas aeruginosa Phosphorylcholine Phosphatase.

Otero LH, Beassoni PR, Boetsch C, Lisa AT, Domenech CE - Enzyme Res (2011)

Bottom Line: However, Zn(2+) is more effective than Mg(2+) at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site.We postulate that Zn(2+) induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure.In contrast, Mg(2+) produces a relaxed or open conformation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional 36 Km 601, Río Cuarto, 5800 Córdoba, Argentina.

ABSTRACT
Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho), is activated by Mg(2+) or Zn(2+), 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 Zn(2+) or Mg(2+) follows a random sequential mechanism. However, Zn(2+) is more effective than Mg(2+) at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn(2+) induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. In contrast, Mg(2+) produces a relaxed or open conformation.

No MeSH data available.


Catalytic mechanism of PchP with Pcho as substrate. The data shown in Figure 1 were analyzed with the program DYNAFIT. E: enzyme; S: substrate, Pcho; A: divalent cation activator; P: product.
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sch1: Catalytic mechanism of PchP with Pcho as substrate. The data shown in Figure 1 were analyzed with the program DYNAFIT. E: enzyme; S: substrate, Pcho; A: divalent cation activator; P: product.

Mentions: In this study, the experimental data treated with DYNAFIT resulted in two schemes for the PchP catalytic mechanism. Scheme 1 illustrates that (i) PchP binds two substrate molecules and one metal ion and (ii) the more probable mechanism for the catalytic action of PchP with Pcho is independent of the identity of the metal ion. Considering the first part of Scheme 1 (top), the kinetic parameters are consistent with a random sequential mechanism in which the metal ion (A) or the substrate (S) initially binds to PchP (E). The EA or ES complexes bind to S or A, respectively, to form the EAS productive complex before the enzyme releases the product (P). The second part of Scheme 1 (bottom) shows a random mechanism for the interaction of a second substrate molecule with the ES or EAS complexes to form SES or SEAS complexes (S before E in SES or in SEAS complexes indicates that the second Pcho molecule binds to a site different from the catalytic site of PchP). After SES binds, the metal ion also forms the SEAS complex, which is capable of forming P but does so with less efficiency than the EAS complex. An interesting comparison can be made between the catalytic mechanisms resulting from the use of the two different substrates, Pcho and p-NPP. Using p-NPP as the substrate, an ordered mechanism occurs in the presence of Mg2+ or Zn2+ [4]. The discrepancy between the catalytic mechanisms may be caused by the different affinity of PchP for the substrates; one of the them is positively charged with an N-trimethylammonium moiety (KM Pcho ≈ 0.020 mM), and the other contains a p-nitrophenol group (KM p−NPP ≈ 3 mM). This difference in affinities may lead to a more equal competition between the metal and substrate for the enzyme because the presence of metal is not required for Pcho binding.


Different Effects of Mg and Zn on the Two Sites for Alkylammonium Compounds in Pseudomonas aeruginosa Phosphorylcholine Phosphatase.

Otero LH, Beassoni PR, Boetsch C, Lisa AT, Domenech CE - Enzyme Res (2011)

Catalytic mechanism of PchP with Pcho as substrate. The data shown in Figure 1 were analyzed with the program DYNAFIT. E: enzyme; S: substrate, Pcho; A: divalent cation activator; P: product.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102446&req=5

sch1: Catalytic mechanism of PchP with Pcho as substrate. The data shown in Figure 1 were analyzed with the program DYNAFIT. E: enzyme; S: substrate, Pcho; A: divalent cation activator; P: product.
Mentions: In this study, the experimental data treated with DYNAFIT resulted in two schemes for the PchP catalytic mechanism. Scheme 1 illustrates that (i) PchP binds two substrate molecules and one metal ion and (ii) the more probable mechanism for the catalytic action of PchP with Pcho is independent of the identity of the metal ion. Considering the first part of Scheme 1 (top), the kinetic parameters are consistent with a random sequential mechanism in which the metal ion (A) or the substrate (S) initially binds to PchP (E). The EA or ES complexes bind to S or A, respectively, to form the EAS productive complex before the enzyme releases the product (P). The second part of Scheme 1 (bottom) shows a random mechanism for the interaction of a second substrate molecule with the ES or EAS complexes to form SES or SEAS complexes (S before E in SES or in SEAS complexes indicates that the second Pcho molecule binds to a site different from the catalytic site of PchP). After SES binds, the metal ion also forms the SEAS complex, which is capable of forming P but does so with less efficiency than the EAS complex. An interesting comparison can be made between the catalytic mechanisms resulting from the use of the two different substrates, Pcho and p-NPP. Using p-NPP as the substrate, an ordered mechanism occurs in the presence of Mg2+ or Zn2+ [4]. The discrepancy between the catalytic mechanisms may be caused by the different affinity of PchP for the substrates; one of the them is positively charged with an N-trimethylammonium moiety (KM Pcho ≈ 0.020 mM), and the other contains a p-nitrophenol group (KM p−NPP ≈ 3 mM). This difference in affinities may lead to a more equal competition between the metal and substrate for the enzyme because the presence of metal is not required for Pcho binding.

Bottom Line: However, Zn(2+) is more effective than Mg(2+) at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site.We postulate that Zn(2+) induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure.In contrast, Mg(2+) produces a relaxed or open conformation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional 36 Km 601, Río Cuarto, 5800 Córdoba, Argentina.

ABSTRACT
Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho), is activated by Mg(2+) or Zn(2+), 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 Zn(2+) or Mg(2+) follows a random sequential mechanism. However, Zn(2+) is more effective than Mg(2+) at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn(2+) induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. In contrast, Mg(2+) produces a relaxed or open conformation.

No MeSH data available.