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 phospholipa se A2 enzymes (hmw-PLA2) have been determined under conditions in whic h hydrolysis of substrate vesicles occurs without the desorption of en zyme from the interface (scooting mode catalysis). The rat kidney enzy me binds to vesicles of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholi ne (OPPC), which contain the substrate stearoyl-2-arachidonyl-sn-glyce ro-3-phosphocholine (SAPC) and 10 mol % arachidonic acid (20:4) and 1- stearoyl-sn-glycero-3-phosphocholine (S-lyso-PC) as the hydrolysis rea ction 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 vesic le binding measurements. The initial rates of hydrolysis of either rad iolabeled -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 inco rporated into product-containing OPPC vesicles, were simultaneously me asured with dual isotope radiometric assays. The plasmenylcholine 1'-e nyl)-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 hydrolys is of the labeled substrates; the rank order of the values is SAPC con gruent-to SAPI congruent-to PlasAPC > SAPE > SAPA congruent-to SAPS. T he maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discrimate between phospholi pids with different polar head groups. The diglyceride 1-stearoyl-2-ar achidonyl-sn-glycerol is not a substrate for the human hmw-PLA2. Two m ixtures of 1-stearoyl-2-acyl-sn-glycero-3-phosphocholine, which have d ifferent sn-2 acyl chains, were prepared and compared to SAPC as subst rates. One mixture contained naturally-occurring unsaturated fatty acy l chains and the other contained a mixture of 20:4, all of its partial ly 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 > s n-2-alpha-linolenoyl > sn-2-linoleoyl > sn-2-oleoyl greater-than-or-eq ual-to sn-2-palmitoleoyl. The preference order of the 20-carbon acyl c hains is 20:4 > 20:3 > 20:2 > 20:1 > 20:0, and there is a preference f or positional isomers with double bonds closest to the sn-2 ester. In contrast, the human non-pancreatic-secreted 14-kDa phospholipase A2 do es not discriminate significantly between the 20-carbon substrates.