PROCESSIVE INTERFACIAL CATALYSIS BY MAMMALIAN 85-KILODALTON PHOSPHOLIPASE-A2 ENZYMES ON PRODUCT-CONTAINING VESICLES - APPLICATION TO THE DETERMINATION OF SUBSTRATE PREFERENCES

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) congruent-to 2 x 10(7) M-1 s-1. Upper limits of k(off) less-than-or-equal-to 3 x 10(-4) s-1 and K(D) less-than-or-equal-to 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 (H-3-SAPS), -phosphoethanolamine (H-3-SAPE), -phosphoinositol (C-14-SAPI), or -phosphate (H-3-SAPA) and either H-3-SAPC or C-14-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 (H-3-PlasAPC) was also tested. Relative substrate specificity constants (k(cat)/K(M)* values) were determined from the concentrations and initial rates of hydrolysis of the labeled substrates; the rank order of the values is SAPC congruent-to SAPI congruent-to PlasAPC > SAPE > SAPA congruent-to SAPS. The maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discrimate 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 greater-than-or-equal-to sn-2-palmitoleoyl. The preference order of the 20-carbon acyl chains is 20:4 > 20:3 > 20:2 > 20:1 > 20:0, and there is a preference for positional isomers with double bonds closest to the sn-2 ester. In contrast, the human non-pancreatic-secreted 14-kDa phospholipase A2 does not discriminate significantly between the 20-carbon substrates.