MUTATION OF AN EF-HAND CA2-BINDING MOTIF IN PHOSPHOLIPASE-C OF DICTYOSTELIUM-DISCOIDEUM - INHIBITION OF ACTIVITY BUT NO EFFECT ON CA2+-DEPENDENCE()

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ i ons for substrate hydrolysis. The role of an EF-hand Ca2+-binding moti f in Ca2+-dependent PLC activity was investigated by site-directed mut agenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residu es with oxygen-containing side chains at co-ordinates x, y, z,-x and - z of the putative Ca2+-binding-loop sequence were replaced by isoleuci ne (x), valine (y) or alanine (z, -x and -z). The mutated proteins wer e expressed in a Dictyostelium cell line with a disrupted pie gene dis playing no endogenous PLC activity, and PLC activity was measured in c ell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2binding, had little effect on Ca2+ affinity and maximal enzyme activit y. 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-maxima l activity was unaltered, but PLC activity levels at saturating Ca2+ c oncentrations were markedly decreased. The results demonstrate that, a lthough the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca2+-dependence of the PLC reaction.