Localization of the second calcium ion binding site in porcine and equine phospholipase A2.
At alkaline pH porcine pancreatic phospholipase A2 is known to bind two Ca2+ ions per protein molecule. One Ca2+ ion is strongly bound to the active site and is essential for enzyme activity. A second Ca2+ ion binds more weakly to the protein and improves the affinity of the enzyme for lipid-water interfaces severalfold at high pH values. A group having a pK around 6 controls enzyme binding to lipid-water interfaces in the absence of Ca2+. By use of proton titration techniques this group is now identified to be a carboxylate having an abnormally high pK. Its pK shifts to a value around 4.5 in the presence of high Ca2+ concentrations, suggesting that the carboxylate is involved in binding the second Ca2+ ion. The carboxylate was identified to be Glu71 by comparing proton titration experiments on porcine pancreatic phospholipase A2 and an isoenzyme. The isoenzyme differs by only four residues from the most abundant enzyme, lacking the carboxylate at position 71 (Asn for Glu). The isoenzyme also appeared to be devoid of an abnormal carboxylate. Identification of Glu71 as the abnormal carboxylate in the porcine enzyme was substantiated by comparison with enzymes from other sources. Kinetic experiments on the various phospholipases finally demonstrated that enzyme species containing Glu71 bind a second Ca2+ ion to the low-affinity site, whereas enzymes lacking Glu71 also lack this second site. These experiments confirm the suggestion that Glu71 is one of the ligands for Ca2+ in the low-affinity site.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006736:Horses; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D011487:Protein Conformation; D013045:Species Specificity; D013552:Swine
D000596:Amino Acids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium