Site-directed mutagenesis of Cys(324) and Cys(331) in human cytosolic phospholipase 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) and Cys(331), chemical modification of which using thiol modifying reagents abolishes the activity of the enzyme [Li et al. (1994) Biochemistry; 33, 8594-8603]. To verify the functional importance of the two cysteine residues, site-directed mutagenesis has been used to create six mutations at positions 324 and 331. The mutant enzymes include C324A, C331A, C324Q, C331Q, C324R, and C331S. Complete loss of activity is observed for C331Q, whereas the other mutants have retained varying degrees of activity. These results show that neither Cys(324) nor Cys(331) is catalytically essential for the enzyme activity. Further chemical modification studies of the mutant enzymes by thiol-specific reagents suggest that modification of Cys(331) is responsible for the complete loss of the enzyme activity, The possible roles of Cys(324) and Cys(331) are discussed.