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)

摘要

pSeveral 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-iM/isubr/sub group I-III phospholipases Asub2/sub (PLAsub2/subs). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLAsub2/subs, 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 PLAsub2/sub from iEchis carinatus sochureki/i (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLAsub2/sub. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Casup2+/sup-binding site of PLAsub2/subs), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaClsub2/sub was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLAsub2/sub. The Casup2+/sup-dependencies of the enzymic activities of ecarpholin S and rPLAsub2/sub were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Casup2+/sup was a prerequisite for this platelet-aggregating effect. Computer modelling of the Casup2+/sup-binding site of Ser-49 PLAsub2/sub compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Casup2+/sup-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 Casup2+/sup binding. Sequence comparisons of ecarpholin S and other low-iM/isubr/sub PLAsub2/subs predicts the presence of a Ser-49 group in the protein family of low-iM/isubr/sub PLAsub2/subs that is distinct from the Asp-49 and Lys-49 groups./p