Serine 228 is essential for catalytic activities of 85-kDa cytosolic phospholipase A2.
The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).
Sharp
J D
JD
Lilly Research Laboratories, Indianapolis, Indiana 46285.
Pickard
R T
RT
Chiou
X G
XG
Manetta
J V
JV
Kovacevic
S
S
Miller
J R
JR
Varshavsky
A D
AD
Roberts
E F
EF
Strifler
B A
BA
Brems
D N
DN
D000595:Amino Acid Sequence; D002384:Catalysis; D016384:Consensus Sequence; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D008245:Lysophospholipase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*
D012694:Serine; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase