Interaction of alkylammonium ions with the active site of P. aeruginosa phosphocholine phosphatase

BEASSONI, PR; OTERO, LH; BOETSCH, C; DOMENECH, CE

Carlos Paz, Cordoba

Congreso; XLIV Reunión Anual de la SAIB; 2008

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Pseudomonas aeruginosa phosphocholine phosphatase (PchP) and hemolytic phospholipase C (PlcH) both catalyze the hydrolysis of compounds containing choline. PlcH on phosphatidylcholine or sphingomyelin produces phosphocholine, substrate of PchP, which is hydrolyzed to choline and inorganic phosphate. Both enzymes recognize the alkylammonium moiety in their respective substrates. Therefore, a parallel study of these two enzymes might help us to find this recognition site in both proteins. To achieve this objective, we started with the study of PchP based in a molecular model which involves the catalytic site formed by motifs I, II, and III (31DMDNT35, 166S, and 261GDTPDSD267, respectively). Kinetic studies were performed with wild type and site directed mutated variants using p-NPP as substrate and Mg2+  as cofactor at pH 5. All the alkylammonium ions tested (simple ones as trimethylamine, choline, betaine, or complex ones as atropine, gallamine, decamethonium, neostigmine, etc.) were inhibitors of PchP. In general, the inhibition contained competitive and noncompetitive components. From motifs I, II and III, only the seryl residue of motif II, S166, seems to be involved in the recognition of the ammonium quaternary moiety. After bioinformatics studies, we concluded that the choline binding domain found in gram positive bacteria or in higher organisms is not present in PchP