The recombinant human 85-kDa cytosolic phospholipase A(2) (cPLA(2)), when assayed in the presence of glycerol, catalyzes the transfer of acyl chains of radiolabeled phosphatidylcholine and parasubstituted phenyl esters of fatty acids to glycerol, in addition to hydrolyzing these substrates. The product of the transacylation reaction is monoacylglycerol (MAG), and the acyl chain is predominantly esterified (greater than or equal to 95%) to a primary hydroxyl group of glycerol (sn-1/3); the stereochemistry is not known. Increasing concentrations of glycerol accelerate enzyme turnover both by providing an additional mechanistic pathway for the enzyme-substrate complex to form products and by increasing the intrinsic hydrolytic and transacylation activities of the enzyme. Significant enzymatic hydrolysis of sn-1/3-arachidonylmonoacylglycerol. was measured, while sn-1/3-alpha-linolenoyl- and sn-2-arachidonylmonoacylglycerols were not detectably hydrolyzed. 1,3-Propanediol also serves as an acyl acceptor for the enzyme. cPLA(2) hydrolyzes an analog of lysophosphatidylcholine that lacks the sn-2 hydroxyl group. The enzyme will hydrolyze sn-1-acyl chains of rac-1-(arachidonyl, alpha-linolenoyl, palmitoyl)-2-O-hexadecyl-glycero-3-phosphocholine lipids and transfer the acyl chain to glycerol. Thus, cPLA(2) has phospholipase A(1) activity but only if an ether linkage rather than an ester linkage is present at the sn-2 position, and it is shown that the sn-1 acyl chains of both enantiomers of phosphatidylcholine are hydrolyzed. Phenyl [C-14]-alpha-linolenate and five para-substituted phenyl esters of [H-3]-alpha-linolenic acid with pK, values ranging from 7.2 to 10.2 for the phenol leaving groups were incorporated into 1,2-ditetradecyl-sn-glycero-3-phosphomethanol/Triton X-100 mixed micelles as substrates for the transacylation/hydrolysis reactions of the enzyme. Average product ratios, which are defined as the amount of monoacylglycerol formed to phenyl ester hydrolyzed, were 2.1 +/- 0.1 (n = 5) for the para-substituted phenyl esters and 2.0 +/- 0.3 (n = 7) for phenyl alpha-linolenate. The similarity of the ratios, despite the range of pK(a) values for the leaving groups, is consistent with the formation of a common enzyme intermediate that partitions to give either fatty acid or MAG. That intermediate may be a covalent acyl enzyme. Finally, the acyl chain specificity of cPLA(2) was investigated to better understand the preference of the enzyme for phospholipids with sn-2-arachidonyl chains.
