Site-Directed Mutagenesis of Cys324 and Cys331 in Human Cytosolic Phospholipase A2: Locus of Action of Thiol Modification Reagents Leading to Inactivation of cPLA2

Abstract

Human cytosolic phospholipase A2 contains two cysteines, cyS324 and cyS331, 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 CyS324 nor CyS311 is catalytically essential for the enzyme activity. Further chemical modification studies of the mutant enzymes by thiol-specific reagents suggest that modification of Cys331 is responsible for the complete loss of the enzyme activity. The possible roles of Cys324 and Cys331 are discussed.