title: Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.
abstract: Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004023:Dictyostelium / Q000201:enzymology*; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D014336:Troponin / Q000737:chemistry; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D017931:DNA Primers; D014336:Troponin; D010738:Type C Phospholipases; D002118:Calcium