Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2.
In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.
van den Bergh
C J
CJ
Department of Biochemistry, State University of Utrecht, The Netherlands.
Bekkers
A C
AC
Verheij
H M
HM
de Haas
G H
GH
D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D005815:Genetic Code; D005971:Glutamates / Q000494:pharmacology*; D008968:Molecular Conformation; D009154:Mutation; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013552:Swine; D014162:Transfection
D005971:Glutamates; D001224:Aspartic Acid; D004247:DNA; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium