Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A(2)

Ser-228 has been shown to be essential for the catalytic activity of the human cytosolic phospholipase A(2) (cPLA(2)). However, its involvement in catalysis has not yet been demonstrated. Using site-directed mutagenesis, active-site directed irreversible inhibitors, and the novel fluorogenic substrate 7-hydroxycoumarinyl gamma-linolenate, evidence is presented to show that the hydroxyl group of Ser-228 is the catalytic nucleophile of cPLA(2), Replacement of Ser-228 by Ala, Cys, or Thr resulted in the inability of these mutants to mediate calcium ionophore induced PGE(2) production in COS-7 cells cotransfected with the cPLA(2) mutants and cyclooxygenase-1, Cell lysates from these transfected cells also had undetectable levels of cPLA(2) phospholipid hydrolyase activity as did the affinity column purified S228A and S228C cPLA(2) mutants overexpressed in insect cells, The loss in activity was not due to the inability of the mutant enzymes to translocate to the substrate lipid interface since the purified S228C cPLA(2) mutant, like the wild type, translocated to the phospholipid membrane in the presence of calcium as judged by fluorescence energy transfer. However, when an activated substrate, 7-hydroxycoumarinyl gamma-linolenate (pK(a) approximate to 7.8 for its leaving group) was used as substrate, there was a significant level of 7-hydroxycoumarin esterase (7-HCEase) activity (about 1% of wild type) associated with the purified S228C cPLA(2) mutant. The S228A cPLA(2) mutant was catalytically inactive, Contrary to wild type cPLA(2), the 7-HCEase activity of the thio-cPLA(2) was not titrated by the irreversible active-site-directed inhibitor methyl arachidonyl fluorophosphonate, but rather titrated by one equivalent of arachidonyl bromomethyl ketone, an arachidonyl binding site directed sulfhydryl reagent. These results are compatible with the hydroxyl of Ser-228 being the catalytic nucleophile of cPLA(2) and that cysteine can replace serine as the nucleophile, resulting in a thiol-cPLA(2) with significantly reduced catalytic power.