Asp-49 is not an absolute prerequisite for the enzymic activity of low-Mr phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper)

Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low-M_r group I-III phospholipases A₂ (PLA₂s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA₂s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA₂ from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA₂. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca²⁺-binding site of PLA₂s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl₂ was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA₂. The Ca²⁺-dependencies of the enzymic activities of ecarpholin S and rPLA₂ were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca²⁺ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca²⁺-binding site of Ser-49 PLA₂ compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca²⁺-binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca²⁺ binding. Sequence comparisons of ecarpholin S and other low-M_r PLA₂s predicts the presence of a Ser-49 group in the protein family of low-M_r PLA₂s that is distinct from the Asp-49 and Lys-49 groups.