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Substrate specificities of the human and rat kidney 85-kDa phospholipase A2 enzymes (hmw-PLA2) have been determined under conditions in which hydrolysis of substrate vesicles occurs without the desorption of enzyme from the interface (scooting mode catalysis). The rat kidney enzyme binds to vesicles of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), which contain the substrate 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) and 10 mol% arachidonic acid (20:4) and 1-stearoyl-sn-glycero-3-phosphocholine (S-lyso-PC) as the hydrolysis reaction products, with a second-order rate constant k(on) approximately equal to 2 x 10(7) M-1 s-1. Upper limits of k(off) < or = 3 x 10(-4) s-1 and KD < = or 15 pM for the dissociation rate and equilibrium constants, respectively, are estimated from the vesicle binding measurements. The initial rates of hydrolysis of either radiolabeled 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphoserine (3H-SAPS), -phosphoethanolamine (3H-SAPE), -phosphoinositol (14C-SAPI), or -phosphate (3H-SAPA) and either 3H-SAPC or 14C-SAPC, which were incorporated into product-containing OPPC vesicles, were simultaneously measured with dual isotope radiometric assays. The plasmenylcholine 1-O-(Z-hexadec-1'-enyl)-2-arachidonyl-sn-glycero-3- phosphocholine (3H-PlasAPC) was also tested. Relative substrate specificity constants (Kcat/KM* values) were determined from the concentrations and initial rates of hydrolysis of the labeled substrates; the rank order of the values is SAPC approximately equal to SAPI approximately equal to PlasAPC > SAPE > SAPA approximately equal to SAPS. The maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discriminate between phospholipids with different polar head groups. The diglyceride 1-stearoyl-2-arachidonyl-sn-glycerol is not a substrate for the human hmw-PLA2. Two mixtures of 1-stearoyl-2-acyl-sn-glycero-3-phosphocholine, which have different sn-2 acyl chains, were prepared and compared to SAPC as substrates. One mixture contained naturally-occurring unsaturated fatty acyl chains and the other contained a mixture of 20:4, all of its partially hydrogenated analogues (20:3, 20:2, and 20:1), and arachidic acid (20:0). The order of preference for the human hmw-PLA2 is sn-2-20:4 > sn-2-alpha-linolenoyl > sn-2-linoleoyl > sn-2-oleoyl > or = sn-2-palmitoyleoyl.(ABSTRACT TRUNCATED AT 400 WORDS)

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The recombinant human 85-kDa cytosolic phospholipase A2 (cPLA2), when assayed in the presence of glycerol, catalyzes the transfer of acyl chains of radiolabeled phosphatidylcholine and para-substituted 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 (> or = 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. cPLA2 hydrolyzes 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, cPLA2 has phospholipase A1 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 [14C]-alpha-linolenate and five para-substituted phenyl esters of [3H]-alpha-linolenic acid with pKa 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 pKa 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 cPLA2 was investigated to better understand the preference of the enzyme for phospholipids with sn-2-arachidonyl chains.