Open AccessGlutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A 2
Author(s) -
BERGH Carel J.,
BEKKERS Augustinus C. A. P. A.,
VERHEIJ Hubertus M.,
HAAS Gerard H.
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14831.x
Subject(s) - calcium , phospholipase , chemistry , biochemistry , mutant , phospholipase c , aspartic acid , binding site , phospholipase a2 , binding domain , phospholipase d , amino acid , enzyme , organic chemistry , gene
In addition to the Ca 2+ ion at the active site, porcine pancreatic phospholipase A 2 (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.