MUTATIONS WITHIN A HIGHLY CONSERVED SEQUENCE PRESENT IN THE X-REGION OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C-DELTA(1)

Phosphoinositide-specific phospholipase C (PI-PLC) enzymes have consid erable structural similarity within limited regions (X and Y) implicat ed in catalysis. The role of residues contained within a highly conser ved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta(1) isoenzyme. Seven residues (Ser-308, Ser-3 09, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residu es, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme ac tivity. The k(cat) of hydrolysis of phosphatidylinositol 4,5-bisphosph ate by H311A and Y314A mutants was reduced 1000- and 10-fold respectiv ely, with little effect on K-m. Further analysis of H311A and Y314A mu tants, using limited proteolysis and circular dichroism, had shown tha t no major structural alterations had occurred. Since site-directed mu tagenesis demonstrated the importance of histidine residues, their rol e in enzyme function was also analysed by chemical modification with d iethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.