The role of Asp-49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity
✍ Scribed by Carel J. van den Bergh; Arend J. Slotboom; Hubertus M. Verheij; Gerard H. de Haas
- Publisher
- John Wiley and Sons
- Year
- 1989
- Tongue
- English
- Weight
- 778 KB
- Volume
- 39
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
The role of aspartic acid49 (Asp-49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp49 is essential for effective hydrolysis of phospholipids. Conversion of Asp49 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 naturally occurring Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivonrs yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself has no enzymatic activity. Our results indicate that Asp49 is essential for the catalytic action of phospholipase A2. The importance of Asp49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The Occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.