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 considerable structural similarity within limited regions (X and Y) implicated in catalysis. The role of residues contained within a highly conserved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta 1 isoenzyme. Seven residues (Ser-308, Ser-309, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residues, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme activity. The kcat of hydrolysis of phosphatidylinositol 4,5-bisphosphate by H311A and Y314A mutants was reduced 1000- and 10-fold respectively, with little effect on Km. Further analysis of H311A and Y314A mutants, using limited proteolysis and circular dichroism, had shown that no major structural alterations had occurred. Since site-directed mutagenesis demonstrated the importance of histidine residues, their role in enzyme function was also analysed by chemical modification with diethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.
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Chester Beatty Laboratories, London, U.K.
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D000595:Amino Acid Sequence; D000828:Animal Population Groups / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005796:Genes; D006639:Histidine / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D004047:Diethyl Pyrocarbonate; D002118:Calcium