SOCIETY FOR SERINE-228 IS ESSENTIAL FOR CATALYTIC ACTIVITIES OF 85-KDA CYTOSOLIC PHOSPHOLIPASE-A(2)

The Ca2+-sensitive cytosolic phospholipase A(2) (cPLA(2)) displays bot h a phospholipase A(2) and a lysophospholipase activity. Numerous hydr olases, including lipases, catalyze the hydrolysis of ester bonds by m eans of an active site triad of amino acids that includes a serine or a cysteine residue, We have examined whether human cPLA(2) belongs to this class of enzymes by using site-directed mutagenesis. Although che mical inactivation of cPLA(2) by the sulfhydryl reagent N-ethylmaleimi de made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA(2) proved dispensable, allowing near-norm al enzyme activity when substituted by alanine. We noted that cPLA(2) contains a 110-amino-acid region with sequence homology to phospholipa se B (PLB) from Penicillium notatum. Interestingly, one of the conserv ed serines of cPLA(2), Ser-228, within this domain aligns with the lip ase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacem ent of Ser-228 by alanine (or threonine or cysteine) yielded catalytic ally inactive cPLA(2), even though the native conformation was maintai ned as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contras t, substitution by alanine of three different serines of cPLA(2) (Ser- 195, Ser-215, or Ser-577) that also aligned with the PLB sequence allo wed for substantial enzymatic activity of cPLA(2). Our findings provid e evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA(2) and that 2) both the phospholipase A(2) and the lysophospholip ase activities of cPLA(2) are catalyzed by the same active site residu e(s).