SITE-DIRECTED MUTAGENESIS OF CYS(324) AND CYS(331) IN HUMAN CYTOSOLICPHOSPHOLIPASE A(2) - LOCUS OF ACTION OF THIOL MODIFICATION REAGENTS LEADING TO INACTIVATION OF CPLA(2)

Human cytosolic phospholipase A(2) contains two cysteines, Cys(324) an d Cys(331), chemical modification of which using thiol modifying reage nts abolishes the activity of the enzyme [Li et al. (1994) Biochemistr y; 33, 8594-8603]. To verify the functional importance of the two cyst eine residues, site-directed mutagenesis has been used to create six m utations at positions 324 and 331. The mutant enzymes include C324A, C 331A, C324Q, C331Q, C324R, and C331S. Complete loss of activity is obs erved for C331Q, whereas the other mutants have retained varying degre es of activity. These results show that neither Cys(324) nor Cys(331) is catalytically essential for the enzyme activity. Further chemical m odification studies of the mutant enzymes by thiol-specific reagents s uggest that modification of Cys(331) is responsible for the complete l oss of the enzyme activity, The possible roles of Cys(324) and Cys(331 ) are discussed.