Aldolase A Ins(1,4,5)P₃-binding domains as determined by site-directed mutagenesis

We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1,4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P₃ binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P₃ binding, increased EC₅₀ values for Fru(1,6)P₂-evoked release of bound Ins(1,4,5)P₃ and increased K₁ values for Ins(1,4,5)P₃-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P₃. Computer-simulated docking of Ins(1,4,5)P₃ in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P₃ to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P₂ binding. Results indicate that Ins(1,4,5)P₃ binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P₃ and Fru(1,6)P₂ for binding. Although Ins(1,4,5)P₃ binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4,5)P₃-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P₃ in other proteins.