Catalysis by phospholipase C delta1 requires that Ca2+ bind to the catalytic domain, but not the C2 domain.
The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC) is absolutely dependent on Ca2+. The PH domain truncated catalytic core of rat phospholipase C delta1 (PLC-delta1) has Ca2+ binding sites in its catalytic and C2 domains, and potential Ca2+ binding sites in two EF-hands. A catalytically inactive PLC-delta1 catalytic core bound with low affinity to PIP2-containing vesicles in the presence of Ca2+. A mutant PLC-delta1 has been engineered which lacks the C2 domain Ca2+ binding site and the surrounding loops known as the jaws. Isothermal calorimetric titration showed four Ca2+ ions bind to the wild-type PLC-delta1 catalytic core in solution but only one binds to the C2 domain jaws deletion mutant. The activity and Ca2+ dependence of wild-type and mutant phospholipase Cs were determined using substrate incorporated in detergent micelles and in large unilamellar vesicles. The activities of wild-type and mutant were identical to each other in both assay systems. Wild-type and the C2 jaws deletion mutant of PLC have Hill coefficients of 1.12-1.16 with respect to [Ca2+]. We conclude that a single Ca2+ bound to the catalytic domain is entirely responsible for the Ca2+ dependence of the basal activity of PLC-delta1.
Grobler
J A
JA
Laboratory of Molecular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580, USA.
Hurley
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D002384:Catalysis; D002850:Chromatography, Gel; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054803:Phospholipase C delta; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat; D002118:Calcium