MULTIPLE ENZYMATIC-ACTIVITIES OF THE HUMAN CYTOSOLIC 85-KDA PHOSPHOLIPASE A(2) - HYDROLYTIC REACTIONS AND ACYL TRANSFER TO GLYCEROL

The recombinant human 85-kDa cytosolic phospholipase A(2) (cPLA(2)), w hen assayed in the presence of glycerol, catalyzes the transfer of acy l chains of radiolabeled phosphatidylcholine and parasubstituted pheny l esters of fatty acids to glycerol, in addition to hydrolyzing these substrates. The product of the transacylation reaction is monoacylglyc erol (MAG), and the acyl chain is predominantly esterified (greater th an or equal to 95%) to a primary hydroxyl group of glycerol (sn-1/3); the stereochemistry is not known. Increasing concentrations of glycero l accelerate enzyme turnover both by providing an additional mechanist ic pathway for the enzyme-substrate complex to form products and by in creasing the intrinsic hydrolytic and transacylation activities of the enzyme. Significant enzymatic hydrolysis of sn-1/3-arachidonylmonoacy lglycerol. was measured, while sn-1/3-alpha-linolenoyl- and sn-2-arach idonylmonoacylglycerols were not detectably hydrolyzed. 1,3-Propanedio l also serves as an acyl acceptor for the enzyme. cPLA(2) hydrolyzes a n analog of lysophosphatidylcholine that lacks the sn-2 hydroxyl group . The enzyme will hydrolyze sn-1-acyl chains of rac-1-(arachidonyl, al pha-linolenoyl, palmitoyl)-2-O-hexadecyl-glycero-3-phosphocholine lipi ds and transfer the acyl chain to glycerol. Thus, cPLA(2) has phosphol ipase 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 este rs of [H-3]-alpha-linolenic acid with pK, values ranging from 7.2 to 1 0.2 for the phenol leaving groups were incorporated into 2-ditetradecy l-sn-glycero-3-phosphomethanol/Triton X-100 mixed micelles as substrat es for the transacylation/hydrolysis reactions of the enzyme. Average product ratios, which are defined as the amount of monoacylglycerol fo rmed 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-li nolenate. The similarity of the ratios, despite the range of pK(a) val ues for the leaving groups, is consistent with the formation of a comm on enzyme intermediate that partitions to give either fatty acid or MA G. 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 chai ns.