The Molecular Nature of the F-actin Binding Activity of Aldolase Revealed with Site-directed Mutants

摘要: We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg → Ala), K107A (Lys R148A had minimal actin activity relative wild type (wt) one mutant, K229A intermediate activity. A mutant with ~4,000-fold reduced catalytic activity, D33S (Asp Ser), normal The aldolase substrates product, fructose 1,6-bisphosphate, 1-phosphate, dihydroxyacetone phosphate, reversed gelling wt F-actin, consistent at least partial overlap actin-binding sites on aldolase. Molecular modeling reveals that we have identified are clustered or around pocket molecule. These data confirm interaction is due binding, they suggest electrostatic interactions between residues, rather than net charge, mediate this Low concentration caused formation high viscosity gel networks, while concentrations resulted solation by bundling filaments, a potential role for enzyme regulation cytoplasmic structure.