Anionic center of porcine pancreatic phospholipase A2

The interaction between porcine pancreatic phospholipase A2 and low-molecular fragments of its substrate -- lecithine was studied using gel-diffusion of the enzyme in lecithin-agarose plates. When the inhibitor was added, a decrease in the magnitude of cleared areas (l/l0) around the depots filled with enzyme solution was observed. A marked decrease in l/l0 in the presence of alpha- and beta-glycerophosphates supported the statement that the cathionic center is a part of the enzyme active site SII. The potent inhibition of phospholipase activity in the presence of phosphocholine, choline, acetylcholine, thiocholine and acylthiocholines suggests the existence of an anionic center SIII in the active site. This suggestion is supported by intensive inhibition of phospholipase activity by certain, aliphatic amines. It was shown that the center is spaced in the direction of the cathionic center. SII. The main contribution to the binding of the cathionic lecithin part ("head") with the anionic center SIII is probably provided by the ion-ionic interactions.     

Solution structure of porcine pancreatic phospholipase A2.

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.     

Optically detected magnetic resonance studies of porcine pancreatic phospholipase A2 binding to a negatively charged substrate analogue

The direct binding of porcine pancreatic phospholipase A2 and its zymogen to 1,2-bis(heptanylcarbamoyl)-rac-glycerol 3-sulfate was studied by optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. The zero-field splittings of the single Trp3 residue undergo significant changes upon binding of phospholipase A2 to lipid. Shifts in zero-field splittings, characterized mainly by a reduction of the E parameter from 1.215 to 1.144 GHz, point to large changes in the Trp3 local environment which accompany the complexing of phospholipase A2 with lipid. This may be attributed to Stark effects caused by the binding of a charged group near Trp3 in the enzyme-lipid complex. The cofactor, Ca2+, which is strongly bound to the enzyme active site, has an influence on the bonding, as reflected by smaller zero-field splitting shifts. A relatively small change in the Trp environment was observed for the interaction of the zymogen with lipid.     

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.     

Hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by porcine pancreatic phospholipase A2

The hydrolysis of small unilamellar vesicles made of dipalmitoylphosphatidylcoline by pancreatic phospholipase A2 has been studied under various conditions of temperature and enzyme and substrate concentration using the following three different experimental protocols. When the enzyme was added to the substrate vesicles after being separately adjusted to the temperature of the experiments hydrolysis occurred instantaneously only in the temperature range where the lipid is known to exist in its gel phase, while above the transition range no hydrolysis occurred. Within the transition range, the time course of hydrolysis was characterized by initial very slow rate of hydrolysis (latency phase) followed by an abrupt increase in the rate after a time tau, which is a complex function of temperature and enzyme to substrate ratio. When an enzyme-substrate mixture was first preincubated below Tm and then temperature jumped to a temperature above or within the transition range, the latency phase was markedly shortened. When the temperature jump was to the transition range, this effect is observed even if Ca2+ is absent in the preincubation mixture. However, instantaneous hydrolysis was observed upon temperature jumping the mixture to a temperature high above Tm only if Ca2+ was present in the preincubation medium. In temperature-scanning experiments, hydrolysis was followed while changing the temperature of the enzyme-substrate mixture continuously. Heating an enzyme-substrate mixture from room temperature resulted in an abrupt onset of hydrolysis when the transition range was approached. These results lead us to conclude that two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicles, which for chemically pure bilayers occurs best in the gel phase. This step is followed by a Ca2+-dependent activation of the initially formed enzyme-substrate complex. The latter step only occurs under conditions where the bilayer possesses packing irregularities and probably involves a reorganization of the enzyme-substrate complex. At least one of these two steps appears to involve enzyme-enzyme interaction.     

NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A2.

Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine, with competitive inhibitors derived from the following general structure: [formula: see text] X and Y are alkyl chains with various 'reporter groups'. The interactions between the inhibitor and the enzyme were localized by comparison of 2-D nuclear Overhauser effect spectra using protonated and selectively deuterated inhibitors, and inhibitors with groups having easily identifiable chemical shifts. These experiments led us to the following conclusions for the phospholipase A2/inhibitor/micelle complex: i) the His48 C2 ring proton is in close proximity to both the amide proton and the methylene protons at the sn-1 position of the glycerol skeleton of the inhibitor, ii) the acyl chain of the inhibitor at the sn-2 position makes hydrophobic contacts near Phe5, Ile9, Phe22 and Phe106; iii) no interactions between the acyl chain at the sn-1 position and the protein could be identified. Comparison of our results on the enzyme/inhibitor/micelle ternary complex with the crystal structure of the enzyme-inhibitor complex shows that the mode of inhibitor binding is similar. However, in several cases we found indications that the hydrophobic chains of the inhibitors can have multiple conformations.     

Hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles by porcine pancreatic phospholipase A2

The interaction between dipalmitoylphosphatidylcholine large unilamellar vesicles and porcine pancreatic phospholipase A2 has been studied under a variety of conditions. It was found that the presence of large unilamellar vesicles inhibits the hydrolysis of small unilamellar vesicles at room temperature, and reaction calorimetric experiments showed that protein-lipid interactions in the absence of Ca2+ occur in the gel state with a stoichiometry of about 40 phospho-lipid molecules/protein-binding site. However, hydrolysis can be induced in the gel state under conditions of osmotic shock. On the other hand, hydrolysis is usually observed within the lipid transition temperature range, but then it occurs only after a latency phase during which the hydrolysis is very slow. The duration of this latency phase reaches a minimum near the phase transition temperature. However, if the enzyme-substrate mixture is heated from low temperatures (continuously or by a temperature jump) to a temperature within the phase transition region, hydrolysis occurs instantaneously. These results are in accordance with the conclusions of the preceding paper (Menashe, M., Romero, G., Biltonen, R. L., and Lichtenberg, D. (1986) J. Biol. Chem. 261, 5328-5333) that effective binding of the enzyme to lipid vesicles occurs relatively rapidly in the gel state and that activation of the enzyme-substrate complex requires the existence of structural irregularities in the lipid bilayer. Although hydrolysis products may have a pronounced effect on the time course of the reaction in the transition range, instantaneous hydrolysis can be induced in the phase transition region in the absence of reaction products by appropriate manipulation of the experimental conditions during which no reaction products are produced. Thus reaction products are not essential for activation of porcine pancreatic phospholipase A2. Furthermore, it is shown that the fraction of lipid hydrolyzed during the latency period is a function of the initial substrate concentration in a manner inconsistent with the proposition that the accumulation of a constant critical fraction of reaction products is the basis for activation. Comparison of the results of this study with those of the preceding paper strongly support the previously proposed reaction scheme.     

Retinol induces platelet aggregation via activation of phospholipase A2

All-trans-retinol induced aggregation of rabbit platelets, and this effect could be inhibited by a cyclooxygenase inhibitor and a thromboxane A2 (TXA2) receptor antagonist, indicating an essential role for endogenously produced TXA2. We found a two-phase arachidonic acid release in retinol-stimulated platelets. The first phase was induced by the action of retinol alone and not inhibited by TXA2 receptor antagonist. The second phase was induced via synergistic action of retinol and initially generated small amount of TXA2, and was inhibited by the antagonist. Moreover, we discussed that the arachidonic acid release may be mediated by the action of phospholipase A2.     

Thermodynamics of the binding of Ca2+ to porcine pancreatic phospholipase A2

The binding of Ca2+ to porcine pancreatic phospholipase A2 was studied by batch microcalorimetry. Enthalpies of binding at 25 degrees C were determined as a function of Ca2+ concentration in buffered solutions at pH 8.0 using both the Tris-HCl and Hepes-NaOH buffer systems. The calorimetric results indicate that protons are released on calcium binding and that in addition to the binding of the active-site calcium, there appears to be weak binding of a second Ca2+. Results from potentiometric titrations indicate that this proton release on binding Ca2+ arises from a change in pK of a histidine(s) functional group. The thermodynamic functions delta G0, delta H0 and delta S0 for calcium binding to phospholipase A2 have been determined. These results are compared with literature data for Ca2+ complex formation with some small molecules and also the protein troponin-C.     

Lipid phase separation mediates binding of porcine pancreatic phospholipase A2 to its substrate

Multilamellar liposomes made of equimolar mixtures of dimyristoyl and distearoylphosphatidylcholine were hydrolysed by porcine pancreatic phospholipase A₂. Ph-stat titration, equilibrium gel filtration and differential scanning calorimetry were used to study respectively the enzymic hydrolysis, the enzyme binding and the lipid phase repartition. We demonstrated that the optimal enzyme activity observed in the region where liquid crystalline and gel lipid phases coexist, is due to a drastic increase of the enzyme binding to its substrate. It is suggested that the border region separating the lipid phases could be a privileged site for enzyme insertion. Increase of lateral compressibility due to coexistence of solid and fluid lipid phases will promote the penetration of the hydrophobic interface recognition site (IRS) of phospholipase A₂ into the lipid matrix whereas the active site, distinct from the IRS will attack its substrate independently of the lipid physical state.     

Interfacial activation of porcine pancreatic phospholipase A(2) studied with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled lipids

The interfacial activation of porcine pancreatic phospholipase A(2) (PLA(2)) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C(12)-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C(12)-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C(6)-NBD-PC, detect latency times of PLA(2) action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C(12)-NBD-PC, a substrate analog, but also of product derivative C(12)-NBD-FA. Ultrafiltration experiments show that C(12)-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C(12)-NBD-PC fluorescence was insensitive to products, whereas C(12)-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C(12)-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C(12)-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C(12)-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield.     

Studies on the role of methionine in porcine pancreatic phospholipase A2.

The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.     

Platelet activation by tetraprenol via stimulation of phospholipase A2 action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.     

Effect of selective chemical modification and CNBr-cleavage at methionine20 in catalytic activity and substrate binding properties of porcine pancreatic phospholipase A2

Porcine pancreatic phospholipase A2 contains 2 methionine (Met) residues located at positions 8 and 20, respectively. Reaction of the enzyme with methyliodide and iodoacetic acid resulted in the selective methylation and carboxymethylation, respectively, of Met20. It was found that porcine pancreatic iso-phospholipase A2, possessing only Met8, was not affected by either modification. Reaction of porcine phospholipase A2 with cyanogen bromide in 0.1 N hydrochloric acid gave rise to cleavage only at Met20. The enhanced reactivity of Met20 compared to that of Met8 is in agreement with the known X-ray structure of phospholipase A2 which shows that Met8 is located in the interior of the protein, while Met20 is at the surface. Both methylation and carboxymethylation of Met20 do not significantly affect catalytic and substrate binding properties of the enzyme. In contrast, the more rigorous cleavage at Met20 by CNBr resulted in the loss of catalytic activity, while substrate and Ca2+ binding was diminished only to a limited extent. Most likely CNBr cleavage at Met20 perturbs the active site despite the fact that the N-terminal fragment Ala1-Hse20 is still bound via the disulfide bridge Cys11-Cys77 to the remainder of the protein. The results obtained strongly suggest that the conformation of the sequences Ala1-Hse20 and/or Asp21-Gly26 are important for the maintenance of the special microenvironment of the active site cleft.     

Interfacial catalysis by phospholipase A2: activation by substrate replenishment.

Polymyxin B (Px), a cyclic cationic peptide, was shown to act as a potent activator of interfacial catalysis by phospholipase A2 (PLA2) acting on dimyristoylphosphatidylmethanol vesicles in the scooting mode. A 7-fold increase in the initial enzymatic velocity was seen with the pig pancreatic PLA2 in the presence of 1 microM Px. Initial experiments including the dependency of the degree of activation by Px on the source of the PLA2 suggested that Px bound to a cationic binding site on the enzyme. However, numerous additional observations led to the conclusion that activation by Px was due to its effects on the substrate interface. For example, the activation by Px was only seen when the PLA2 acted on small vesicles rather than larger ones, and all of the available substrate was eventually hydrolyzed in the presence of a small mole fraction of Px. Px did not promote the intervesicle exchange of PLA2, and it did not alter the binding of the evidence led to the conclusion that Px activated interfacial catalysis by promoting the replenishment of substrate in the enzyme-containing vesicles. When PLA2 was acting on small vesicles in the scooting mode, the observed initial velocity was lower than that measured with large vesicles because the surface concentration of substrate decreased relatively rapidly in the small vesicles. Px promoted the transfer of phospholipids between the vesicles and functioned as an activator by keeping the mole fraction of substrate in the enzyme-containing vesicles close to 1. This effect of Px was consistent with the ability of polycationic peptides to induce the intervesicle mixing of anionic phospholipids in vesicles [Bondeson, J., & Sundler, R. (1990) Biochim. Biophys. Act 1026, 186-194]. Activation by substrate replenishment was quantitatively predicted by the theory of interfacial catalysis on vesicles in the scooting mode. The role of substrate replenishment in the kinetics of interfacial catalysis in phospholipid micelles was discussed. Finally, the protocols developed in this paper were outlined in view of their utility in the analysis of activators of interfacial catalysis.     

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor

Summary The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the -amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.     

Kinetic behavior of porcine pancreatic phospholipase A2 on zwitterionic and negatively charged single-chain substrates

The kinetic properties of porcine pancreatic phospholipase A2 were studied on a series of n-acylglycollecithins and n-acylglycol sulfates containing acyloxy or acylthio ester bonds at substrate concentrations below and above the critical micelle concentration. These single-chain detergents containing a primary (thio) ester bond are hydrolyzed rather slowly by the pancreatic enzyme, and maximal activity was found always for the n-octanoyl derivatives. The acylthio ester group is split 4-5 times faster than the corresponding acyloxy ester function. The kinetic behavior of the enzyme acting on zwitterionic glycollecithins or on anionic glycol sulfates is quite different and provides an explanation for the differences in pH optimum. Both for glycollecithins and for glycol sulfates, maximal enzyme activities are found in high molecular weight aggregates consisting of several enzyme molecules and detergent monomers. Their pathway of formation, however, is not the same.     

Kinetic behavior of porcine pancreatic phospholipase A2 on zwitterionic and negatively charged double-chain substrates

A number of isomeric diacylglycerophosphocholines and diacylglycero sulfates containing O-acyl and/or S-acyl ester bonds were investigated as substrates for porcine pancreatic phospholipase A2 and its zymogen. A comparison is made with the kinetic properties of the enzyme toward the corresponding glycol detergents previously described [van Oort, M. G., Dijkman, R., Hille, J. D. R., & de Haas, G. H. (1985) Biochemistry (preceding paper in this issue)]. Hydrolysis of the secondary ester bond in the 1,2-diacylglycero-3-type lipids proceeds much faster than the splitting of the primary ester function present in the isomeric 1,3-diacylglycerol and 1-acylglycol derivatives. In sharp contrast to the glycol detergents, the substitution of the cleavable oxygen ester by a thio ester bond in the glycerol lipids results in 5 times lower kcat values. At alkaline pH and above the critical micelle concentration, the anionic sulfates are much better substrates than the corresponding phosphocholine-containing detergents. At very low detergent concentrations, below the critical micelle concentration, the anionic sulfates induce protein aggregation such that phospholipase A2, as well as its zymogen, is present in high molecular weight complexes containing several protein molecules. In these aggregates, protein-protein and/or lipid-protein interactions strongly activate phospholipase but not the zymogen.     

The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49]phospholipase A2 from Agkistrodon piscivorus piscivorus' venom

In order to probe the role of Asp-49 in the active site of porcine pancreatic phospholipase A2 two mutant proteins were constructed containing either Glu or Lys at position 49. Their enzymatic activities and their affinities for substrate and for Ca2+ ions were examined in comparison with the native enzyme. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions in particular for the lysine mutant but the affinity for substrate analogues is hardly affected. Extensive purification of [Lys49]phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein which was 4000 times less active than the basic [Asp49]phospholipase A2 from this venom. Inhibition studies with p-bromophenacyl bromide showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself is inactive. The results obtained both with the porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2.