CLONING AND IDENTIFICATION OF AMINO-ACID-RESIDUES OF HUMAN PHOSPHOLIPASE C-DELTA-1 ESSENTIAL FOR CATALYSIS

In vitro single point mutagenesis, inositol phospholipid hydrolysis, a nd substrate protection experiments were used to identify catalytic re sidues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library, Invariant ami no acid residues containing a functional side chain in the highly cons erved X region were changed by in vitro mutagenesis. Most of the mutan t enzymes were still able to hydrolyze inositol phospholipid with acti vity ranging from 10 to 100% of levels in the wild type enzyme. Except ions were mutants with the conversion of Arg(398) to Leu (R338L), Glu( 341) to Gly (E341G), or His(356) to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defectiv e mutant forms of PLC delta 1 could specifically bind to phosphatidyli nositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wi ld type enzyme. Western blotting analysis of trypsin-treated enzyme-PI P2 complexes revealed that a 67-kDa major protein fragment survived tr ypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC de lta 1 was preincubated with 7.5 mu M PIP2, whereas if it was preincuba ted with 80 mu M PIP2, the size of major protein surviving was compara ble to that of intact enzyme. However, mutant enzyme R338L was not pro tected from trypsin degradation by PIP2 binding. These observations su ggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu(541), and His(356) a re not involved in either high affinity or low affinity PIP2 binding b ut rather are essential for the Ca2+-dependent cleavage activity of PL C.