The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49]phospholipase A2 from Agkistrodon piscivorus piscivorus' venom.
In order to probe the role of Asp-49 in the active site of porcine 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 comparison with the native enzyme. Enzymatic characterization 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 from 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 porcine pancreatic phospholipase A2 mutants and with the native venom enzymes show that Asp-49 is essential for the catalytic action of phospholipase A2.
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000502:physiology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003435:Crotalid Venoms / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009154:Mutation; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013552:Swine
D003435:Crotalid Venoms; D010743:Phospholipids; D011994:Recombinant Proteins; D001224:Aspartic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium