The role of aspartic acid‐49 in the active site of phospholipase A2

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Abstract
In order to probe the role of Asp-49 in the active site of procine 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 comparision with the native enzyme. Enzymatic charaterization 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 form 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 procine pancreatic phospholipase A2 mutant with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2.

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