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The aspartyl residue at position 49 in phospholipases A2 (PLA) has been viewed as a component of the catalytic apparatus because of its involvement in binding the essential cofactor, calcium. We recently discovered a new class of PLA's in which, among other changes in highly invariant residues, Asp-49 is replaced by a lysine (Maraganore et al. (1984) J. Biol. Chem. 259, 13839). These Lys-49 PLA's are also calcium-dependent, but, in contrast to the Asp-49 enzymes, they bind phospholipid strongly in the absence of calcium. Lys-49 PLA's are, therefore, ideal for studying structural and mechanistic aspects of these enzymes. Attempts to modify Lys-49 with the amino group-specific reagent, trinitrobenzenesulfonic acid (TNBS) led to the inactivation of the PLA, but reaction occurred not as expected at position 49, but at Lys-53. These findings lead us to propose a model, applicable to PLA's in general, in which cationic side chains at position 53 in these enzymes participate in phospholipid binding on the path to formation of the catalytic complex. This model serves to explain a number of unresolved observations in the current literature relating to enzyme-substrate interactions in the PLA's.

The basic Lys-49 phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus venom is homologous to the basic Asp-49 PLA2 from the same venom as well as other snake venom PLA2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent Vmax of the Lys-49 PLA2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA2, compared to the Asp-49 PLA2 had less than 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA2. Evaluation of the present results, along with earlier findings using Asp-49 PLA2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: The A. p. piscivorus Asp-49 PLA2 enzyme resembles the Asp-49 enzymes from N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. In contrast to some previous suggestions, basic PLA2 enzymes are not necessarily more toxic than neutral or acidic enzymes. Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA2 enzymes exert effects independent of phospholipid hydrolysis.

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.

We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.